A critical evaluation of loss of heterozygosity detected in tumor tissues, blood serum and bone marrow plasma from patients with breast cancer

Blood serum of BCa patients with primary breast cancer (n = 40, stage M0) was taken after surgery before the initiation of adjuvant therapy. Blood serum of patients with metastatic disease (n = 48, stage M1) was collected 1 to 11 years after surgery of the primary tumor. Of 28 patients (n = 12 M0, n = 16 M1), repeated blood collections during the course of treatment were included. In addition, primary tumors and BM aspirates were available from 60 (n = 30 M0, n = 30 M1) and 22 patients (n = 22 M0), respectively. Blood samples were taken between February 2002 and October 2003 from patients who received systemic therapy at the outpatient clinic of the department of gynecology and who agreed to participate in this study.

Patients treated for primary BCa underwent surgery including lumpectomy and dissection of axillary lymph nodes or modified radical mastectomy. All patients had epithelial BCa, and metastatic spread was excluded by chest radiology, liver ultrasound scan and bone scan. These patients received adjuvant treatment (endocrine treatment and/or anthracycline-containing chemotherapy as well as radiotherapy) according to national guidelines.

Patients with metastatic disease received chemotherapy, endocrine treatment, or treatment with the humanized anti-HER2 antibody Trastuzumab (Herceptin) alone or in combination with chemotherapy.

The median age of M0 and M1 patients was 58 (range 29 to 77) and 59 (range 31 to 83) years, respectively.

All patients gave their informed consent and the examination of the clinical material was approved by the local ethics review board.

Sections of 5 μm thickness were cut from formalin-fixed, paraffin-embedded breast tumor blocks and mounted on microscope slides.

Paraffin-embedded sections of the primary tumors were stained for the expression of HER2 (antibody CB 11; Novocastra, Newcastle upon Tyne, UK), estrogen receptor (antibody NCL-6F11, Novocastra), progesterone receptor (antibody NCL-PGR-312, Novocastra) and the proliferation antigen Ki-67 (clone MIB-1; Dako, Hamburg, Germany). Staining for HER2 was expressed as 'DAKO-Score' with values between 0 and 3+ (only 3+ was considered as positive); staining for estrogen and progesterone receptors was evaluated according to the system of Remmele and Stegner [28], and a score greater 1 was regarded as positive. Proliferation was evaluated as the percentage of cells stained positive with the antibody MIB-1.

For microscopic evaluation the paraffin slices were stained with haematoxylin and eosin (Merck, Darmstadt, Germany). The tumor tissue of paraffin slices were either scraped off from H/E-stained slides under a microscope or computer-assisted microdissected from unstained slides via laser pressure catapultation.

Approximately 10 ml blood were drawn by vein puncture and centrifuged at 2,500 g for 10 min. The upper phase contained the blood serum, from which 2 to 3 ml was removed for the extraction and analysis of the circulating DNA. The remaining 17 to 18 ml blood were supplemented up to 50 ml with lysis buffer containing 0.3 M sucrose, 10 mM Tris-HCl pH 7.5, 5 mM MgCl₂ and 1% Triton ×100 (Sigma, Taufkirchen, Germany). Following incubation for 15 min on ice, the isolation and purification of the leukocytes were carried out by two centrifugation steps at 2500 g, 4°C for 20 min.

Genomic DNA was extracted from microdissected tumor tissues, blood serum, BM plasma and leukocytes (reference DNA) of each patient using the QIAamp DNA Mini Kit and a vacuum chamber (Qiagen QIAvac24) according to the manufacturer instructions (Qiagen, Hilden, Germany). Quantifications and qualities of the isolated DNA were spectrophotometrically determined at 260 and 280 nm, respectively (Eppendorf, Hamburg, Germany). Dilution experiments to determine the lowest portion of tumor-specific DNA that could be detected were performed. For this study we mixed and amplified known quantities and proportions of normal (leukocyte) DNA and tumor or plasma DNA.

10 ng of DNA samples were amplified in a 10-μl reaction volume containing PCR Gold buffer (150 mM Tris-HCl, pH 8.0 and 500 mM KCl), 2.5 mM MgCl₂ (Applied Biosystems, Mannheim, Germany) 200 μM dNTPs (Roche, Mannheim, Germany), 11 pM of primer sets (Sigma, Taufkirchen, Germany) and 1.5 U AmpliTaq Gold DNA-Polymerase (Applied Biosystems). The sense primers were fluorescence-labeled (Hex, FAM or TAMRA) at the 5'end. The reaction was started with activation of the DNA polymerase for 5 min at 95°C followed by 40 cycles of PCR amplification. To verify the microsatellite alterations, the experiments were done as duplicates and each PCR was repeated at least twice.

We chose the following microsatellite primers for the current analyses: D3S1255, D9S171, D10S1765, D13S218, D16S421, D17S250 and D17S855, because they are most informative for the particular gene loci and binding to known tumor-suppressor genes [12‐16].

0.5 μl of each PCR product was mixed with 40 μl HiDi Formamid and 0.2 μl 500-ROX size marker (Applied Biosystems, Darmstadt, Germany), which served as an internal standard. Each sample was denatured by heating to 94°C for 2 min. Capillary electrophoresis analysis was performed on a Genetic Analyzer 310 (Applied Biosystems). Samples were injected at 15 kV for 3 s and separation was performed at 15 kV and 60°C. Data were collected with data-collection software and analyzed by Gene-Scan 3.1 software. GeneScan-500 standard fragments in the size range of 100 to 300 bp were used for the calculation of relative sizes of microsatellite alleles. The incidence of LOH was calculated by the ratio of the intensities of the two alleles in tumor tissue, serum and BM plasma in compared with leukocytes. LOH was interpreted if the final quotient was < 0.6 or >1.67. Homozygous and non-analyzable peaks were designated as non-informative cases.

The concentration of DNA in serum of 88 BCa patients was spectrophotometrically quantified, and revealed a range of DNA yields between 70 and 5,000 ng/ml, with a mean value of 500 ng/ml. There was no difference in concentrations of serum DNA between stages M0 and M1. The range of DNA concentrations in the 22 BM samples was between 80 and 800 ng/ml with a mean value of 310 ng/ml, and consequently lower than the DNA levels in serum. Compared with the higher DNA concentrations in blood and BM plasma of BCa patients, healthy women had minor levels of DNA in their serum. The mean value of the DNA concentrations of serum from 10 control women was 63 ng/ml. Repeated blood collections during 3 months showed a constantly low level of DNA in these samples and no occurrence of LOH on the free DNA (data not shown).

Additional file 1 outlines the pattern of LOH detected in primary tumors, blood and BM of each BCa patient with primary (n = 40, stage M0) and metastatic disease (n = 48, stage M1) using a panel of 7 different polymorphic microsatellite markers. Blood serum of the 40 M0 patients was taken after removal of the primary tumor.' Tumor tissues and BM aspirates were available from 30 and 22 of these patients, respectively. Primary tumors from 30 of the 48 M1 patients were obtained at the time of primary surgery, when these patients displayed no clinical signs of overt metastases (that is, 1–11 years before blood sampling). As shown in Additional file 2, 30% of the BCa patients (M0 and M1) harbored at least one LOH at any of the markers tested in their blood serum. Figure 1 shows such a LOH on free serum DNA recorded at the marker D17S855. By contrast, 60% of the patients displayed LOH in their primary tumor, while LOH was detected in the BM plasma in only 18% of these patients.

The LOH index (number of LOH of each patient divided by the informative cases) of the respective patients was higher in primary tumors (27.5%) than in serum (9.0%) and BM (5.0%) samples. Almost half (47%) of the LOHs at any of the markers recorded in serum could be retrieved in the corresponding primary tumors. The detected LOHs in BM could be recovered in 40% and 20% of the matched tumor and blood samples, respectively. Among the informative cases, the frequency of LOH in the tumors was highest for the markers D13S218 (38.0%), D17S855 (36.0%) and D17S250 (33.5%) (Figure 2a). The marker D3S1255 was the predominantly affected region in M0 (19%) and M1 (24%) blood serum, as well as in BM plasma (19%), indicating the same incidence of LOH on serum and BM DNA from M0 patients and a similar rate in the other clinical samples (Figure 2a,b). The high frequencies of LOH at the markers D10S1765 (18.0%), D13S218 (38.0%), D16S421 (29.0%), D17S250 (33.5%) and D17S855 (36.0%) found in tumors could not be retrieved in M0 serum samples (Figure 2a). A comparison of the numbers of LOH recorded in both sera from M0 and M1 patients in Figure 2b shows the increase of the rate of LOH at the markers D3S1255, D10S1765, D13S218 and D16S421 from M0 to M1 stage. At the other markers (D9S171, D17S250 and S17S855) the frequency of LOH was even higher in M0 than M1 serum. The overall rate of LOH in M0 and M1 serum were 10.0% and 8.5%, respectively.

To obtain information on the frequency of LOH during the course of adjuvant chemotherapy and treatment, serial examinations were performed using 1–3 repeated serum samples from 28 BCa patients (n = 12 M0, n = 16 M1). These patients were randomly chosen from the entire BCa patient cohort. In general, the patterns of the repeated blood collections showed no significant differences (Additional file 3).

Of the M0 patients, two women showed LOH in their first serum sample that which disappeared during the adjuvant therapy. By contrast, in serum of two further M0 patients with an initial LOH-negative result, LOH could only be observed in the second and third serum samples (Additional file 3).

The M1 serum samples displayed more frequent LOH during treatment than M0 serum. Whereas two women of the cohort showed LOH in their first serum sample which disappeared during therapy, four patients were initially LOH-negative, and LOH was only detected during therapy in the second and/or third serum samples (Additional file 3).

To determine whether a correlation exists between clinical parameters of the BCa patients and LOHs detected at the different microsatellite markers, we compared tumor size (T1, T2, T3/4), nuclear grade (G1, G2, G3), axillary lymph node status (positive/negative), estrogen receptor (positive/negative), progesterone receptor (positive/negative), the proliferation antigen Ki-67 (low ≤30%/high > 30%) and HER2 expression (positive/negative) with the occurrence of LOH in the different clinical samples. In Additional file 4, the statistically significant associations of LOH profiles detected in primary tumors and blood serum samples with the clinical data are summarized. In the tumor samples, several significant correlations could be detected. The occurrence of LOH at three markers (D3S1255, D9S171 and D17S855) was significantly associated with higher nuclear grade of tumors. At the marker D17S250 LOH was associated with negative estrogen and progesterone receptor status and the high expression of the proliferation antigen Ki-67. Besides its association with the nuclear grade, the marker D9S171 also showed a correlation with the antigen Ki-67 (Additional file 4). Interestingly, in M0 blood serum samples a correlation of LOH at the locus D3S1255 to positive axillary lymph node status was observed (Additional file 4, P = 0.05). Only M0 patients with a positive lymph node status harbored LOHs at this marker. Also, both M0 and M1 blood serum samples together showed a significant correlation of positive axillary lymph node status to LOH at this marker (Additional file 4, P = 0.004).