Lipophilin B: A gene preferentially expressed in breast tissue and upregulated in breast cancer

Abstract

Lipophilin B (LPB), which is also known as BU101, is a secretoglobin which exists in vivo as a complex with the mammary-specific protein, mammaglobin A (MGA). The aim of our study was to investigate the expression of LPB in a panel of breast and nonbreast tissues and compare its expression with that of MGA. Using RT-PCR, LPB mRNA was detected in 16/25 (64%) of normal breast specimens, 23/30 (77%) of fibroadenomas, 102/156 (65%) of primary breast cancers and in 8/36 (22%) nonbreast tissues. Levels of expression of LPB mRNA were significantly higher in breast cancers compared to both normal breast tissues (p = 0.02) and nonbreast tissue (p < 0.001). In the primary breast cancers, expression of LPB mRNA was positively correlated with the estrogen receptor (p = 0.045) but inversely related to both tumor grade (p < 0.001) and proliferation rates (p = 0.0345). Compared to MGA, expression of LPB was more sensitive but less specific for breast cancer. Using Western blotting, LPB migrated with an approximate molecular mass of 7–8 kDa, the expected molecular mass of free LPB. Immunohistochemical analysis of breast cancers showed that LPB expression was predominantly confined to the cytoplasm of tumor cells. We conclude that expression of LPB is preferentially but not exclusively restricted to breast tissue. Since LPB was expressed relatively specifically in breast tissue and was significantly upregulated in breast carcinomas, it is a promising candidate biomarker for breast cancer. © 2006 Wiley-Liss, Inc.

Lipophilin B (LPB) is a member of the secretoglobin supergene family. Currently, at least 23 members of this supergene family are known to exist, 6 of which have been found in humans.1 The human members include, mammaglobin A (MGA), mammaglobin B (also known as lipophilin C), lipophilin A, lipophilin B (also known as BU101), Clara cell 10 kDa protein and the recently identified interferon-gamma-inducible secretoglobin.1, 2 All 6 human genes are located in a dense cluster on chromosome 11q12.2.3 As the name implies, the secretoglobins encode secreted globular proteins. These proteins have low molecular weight and exist in vivo as either homo or heterodimers.

Of the 6 human secretoglobins, MGA is the most widely studied (for review, see Ref.4). Expression of this gene is largely but not exclusively confined to breast tissue.5, 6, 7, 8, 9, 10 Compared to normal breast tissue, expression of MGA in breast carcinomas is upregulated at least 10-fold compared to nonmalignant breast tissue.11 Because its expression is almost exclusive to breast tissue and it is significantly upregulated in breast malignancy, MGA is currently one of the most promising candidate biomarkers for breast cancer.

In breast tissue, MGA exists as a complex with LPB.12 Like MGA, LPB is also a low molecular weight peptide, i.e., with a predicted molecular mass of 7.7 kDa.12 Also, similar to MGA, preliminary findings suggest that the expression of LPB is largely but not exclusively restricted to breast tissue.7, 8, 9, 12, 13 Compared to MGA however, relatively little work has been carried out on LPB. Since LPB is also a potential biomarker for breast cancer, the aim of this investigation was to carry out a detailed study on its expression in normal, benign and malignant breast tissue. For comparison, we also investigated the expression of LPB in a panel of normal and malignant tissues other than breast.

ELISA, enzyme linked immunosorbent assay; ER, estrogen receptor; GAPDH, glyceraldehyde phosphate dehydrogenase; LPB, lipophilin B; MGA, mammaglobin A; PCNA, proliferating cell nuclear antigen; PR, progesterone receptor; RT-PCR, reverse transcriptase-polymerase chain reaction.

Material and methods

Tumor specimens and patients

Table I summarizes the characteristics of the 156 breast cancers analyzed for LPB mRNA expression. The normal breast samples included tissue remote from primary carcinomas (n = 16), remote from fibroadenomas (n = 3) and reduction mammoplasty specimens (n = 6). As similar proportions of these tissues expressed LPB, they were all combined and regarded as “normal” breast tissue. It should be noted however, that these so-called “normal” breast tissues are not from normal, healthy women, which for ethical reasons cannot be obtained. Table II lists the nonbreast tissues investigated. All tissues were obtained from patients undergoing surgery at St Vincent's University Hospital, Dublin. After surgical resection and pathological assessment, tissues were snap-frozen in liquid nitrogen and then stored at −80°C. Tissue homogenization was carried out using a Braun Mikro Dismembrator (Braun, Melsungen, Germany). Part of the powder was extracted with 50 mM Tris-HCl (pH 7.4) containing 1 mM monothioglycerol and used for the assay of estrogen receptor (ER) and progesterone receptor (PR). The residual powder was extracted for total RNA using the guanidinium thiocyanate method.14

Table I. Characteristics of the 156 Primary Breast Cancers Used for RT-PCR of LPB mRNA

Tumour characteristic	n	%
ER, estrogen receptor; PR, progesterone receptor.
Size (cm)
≤2	40	26
>2	102	65
Unknown	14	9
Grade
1 and 2	52	33
3	75	48
Unknown	29	19
Nodes
Negative	65	42
Positive	75	48
Unknown	16	10
ER
Negative	58	37
Positive	83	53
Unknown	15	10
PR
Negative	92	59
Positive	44	28
Unknown	20	13
Histological type
Ductal	119	77
Lobular	20	12
Ductal and lobular	5	3
Unknown	12	8

Table II. Normal and Malignant Tissues Other than Breast Investigated for LPB mRNA (n = 36)

Non-breast normal tissue	n	Non-breast malignant tissue	n
PS, papillary serous; BCC, basal cell carcinoma.
Ovary	1	Ovary (PS)	2
Thyroid	4	Thyroid	1
Skin	3	Skin (BCC)	4
Colon	3	Colon	2
Liver	2	Liver	1
Prostate	1	Prostate	2
Parotid	1	Parotid	2
Ileum	2
Spleen	1
Gallbladder	1
Synovium	2
Muscle	1

Standard RT-PCR for LPB mRNA

Standard RT-PCR of LPB was carried out as previously described.8 In brief, 1 μg of total RNA was reverse transcribed to cDNA in a reaction mixture containing 0.5 mM of each deoxynucleotide triphosphate (dNTP), 10 μg/ml of oligo (dT), 10 mM dithiothreitol, 50 mM Tris-HCl (pH 8.3), 75 mM KCl and 3 mM MgCl₂. The reaction mix was incubated for 5 min at 70°C to remove secondary RNA structures, centrifuged and cooled on ice. Eight units (U) of recombinant human placental ribonuclease inhibitor (Promega, WI, USA) and 200 U of Moloney murine leukemia virus reverse transcriptase (Gibco, Gaithersburg, MD, USA) were then added followed by incubation for a further 60 min at 37°C. Finally, the samples were heated for 5 min at 65°C and then stored at −20°C until required for PCR amplification. Amplification of cDNA was carried out using the following primers sequences: sense, 5′-CAC TCA TTG TTT GTG AAA GCT G-3′ and antisense 3′-GAC AGT GGA AAC CAG GAT GA-3′.

PCR was performed in 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton® X-100, 0.2 mM of each dNTP, 20 pmol each of upstream and downstream primers (Genosys, Pampisford, UK), 2 μl of cDNA, 1.5 mM MgCl₂ and 1 U of Taq DNA polymerase (Promega) in a final volume of 25 μl. All PCR reactions were performed in an automated thermocycler (MJ Research, Watertown, MA). The amplification conditions were as follows: 30 cycles of 30 sec at 94°C, 1 min at 60°C and 1 min at 72°C followed by 5 min at 72°C.

Using these conditions, amplification products were obtained in the exponential phase. After amplification, 10 μl of PCR product was run on a 2% agarose gel. The gels were stained with ethidium bromide and visualized under UV light. As a control, PCR with primers specific for the housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH), was carried out on each sample. RNA isolated from MDA-MD-415 breast cancer cells was used as a positive control. Negative controls included: (a) omission of reverse transcriptase and (b) replacement of cDNA by water. The identity of the LPB PCR products was confirmed by direct sequencing using the ABI Prism 310.

Real-time PCR for LPB mRNA

Real-time PCR using the LightCycler (Roche Molecular Biochemicals, Germany) was also used to determine LPB mRNA expression in the breast cancer cell lines. The primer sequences used were as follows: sense, 5′-CCC TCT GCT GCT ACC A-3′ and antisense 5′-ACC AGG ACT TCC GCA A-3′. Each reaction contained 200 ng cDNA, 3 mM MgCl₂, 40 ng of each sense and antisense primer and 2 μl of LightCycler FastStart reaction Master Mix (containing FastStart Taq DNA polymerase and DNA double-strand specific SYBR Green 1 dye) (Roche Molecular Biochemicals, Mannheim, Germany). The amplification conditions for LPB were as follows: 94°C for 10 min, followed by 35 cycles of 94°C for 10 sec, 50°C for 5 sec and 72°C for 10 sec.

During the optimization phases, PCR products were separated by gel electrophoresis to confirm their identity and size. This is also allowed for the identification of any nonspecific products or primer-dimers. Ten μl of the PCR product was added to 5 μl Blue/Orange loading dye (Promega) and run on a 2% agarose gel along with a 100 bp ladder (Promega). As an internal control, PCR with primers specific for the housekeeping gene, GAPDH, was carried out on each sample. The following primer sequences were used for GAPDH: sense, 5′-GCC TCA AGA TCA TCA GCA A-3′ and antisense, 5′-CCA GCG TCA AAG GTG GAG-3′. The annealing temperature was 64°C. RNA isolated from the ZR-75-1 breast cancer cell line was used as a positive tissue control.

Western blot analysis

Approximately 200 mg of tissue powder was suspended in 400 μl of 50 mM Tris-HCl (pH 7.4), aspirated through a 19-guage needle, vortexed and agitated on ice for 30 min (Gyro-Rocker, Stuart Scientific, England). This was followed by the addition of 0.1% Triton® X-100 (BDH, Poole, England) and a further agitation on ice for 60 min. Samples were then centrifuged at 9,500g (Biofuge fresco, Kendro Laboratories Products) for 20 min at 4°C. The supernatants containing the protein were removed and the protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce).

For Western blotting, 100 μg of total protein was diluted in 4 μl of 0.35 M SDS sample buffer containing 20% β-mercaptoethanol and heated at 95°C for 5 min. Samples were separated by SDS-PAGE electrophoresis using 4–20% gradient gels (Novex Experimental Technology, Frankfurt, Germany). Samples were electrophoresed in SDS-running buffer at a constant voltage of 100 V for approximately 90 min.

The separated protein was transferred to a nitrocellulose membrane (Sigma, Poole, England) using a semidry blotting apparatus (Atto, Tokyo, Japan). Proteins were transferred from the gel onto the nitrocellulose membrane for 45 min at a constant current of 250 mA. Finally, membranes were blocked for 1 hr with 5% (w/v) Marvel (instant dried skimmed milk) in tris buffered saline (TBS) containing 0.05% Triton® X-100 (TBS-T) with continuous shaking at room temperature. Membranes were probed overnight with LPB antibody (H85C21) at a concentration of 2 μg/ml. This is a mouse monoclonal antibody against LPB and was a generous gift from Dr. P. Hemken, Abbott Diagnostics, N. Chicago.

Membranes were washed 3 times for 10 min in TBS-T and incubated with a HRP-conjugated secondary antibody (1:2,000 dilution) (Sigma), for 1 hr with continuous shaking at room temperature. Membranes were again washed 3 times in TBS-T, followed by a 10-min wash in TBS. HRP was detected using luminol chemiluminescence reagent (SantaCruz, Heidelberg, Germany). Chemiluminescence was detected by autoradiography using X-ray film (Fuji, Japan). Concentrated serum-free medium from ZR-75-1 breast cancer cells was used as a positive control while an extract of nonmalignant colon tissue was used as a negative control. A further negative control involved omission of the primary antibody. Beta-actin was used as an internal control.

Immunohistochemistry

Immunohistochemical staining for LPB was carried out on 22 formalin-fixed paraffin-embedded tissue sections of invasive breast tumors. These samples were separate from those used for Western blotting. Sections were deparaffinised in xylene for 10 min, followed by rehydration in 100% methylated spirits (MS) for 10 min and 95% MS for 5 min. After rehydration, sections were incubated in deionized water for 5 min. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 7 min in a humidity chamber.

Prior to staining, heat induced antigen retrieval was performed. Sections were placed in cold 10 mM citrate buffer (pH 6.0) and heated for 6 min until boiling in a microwave oven (Sanyo, Super Showerwave, 800 W). This was followed by 3 cycles at medium power for 5 min each. The citrate buffer containing the sections was allowed to cool for 18 min at room temperature before being washed in phosphate buffered saline (PBS) and blocked with normal horse serum (Vector Laboratories, Peterborough, England) for 20 min. Sections were incubated with the H85C21 monoclonal antibody at a concentration of 0.25 μg/ml in ChemMate™ Antibody Diluent (Dako CytoMation, Glostrup, Denmark) for 1 hr at room temperature.

After a 5-min wash in PBS containing 0.1% Tween 20 (PBS-T), slides were incubated for 20 min with the biotinylated mouse secondary antibody (1:500 dilution in PBS) (Vector Laboratories, Peterborough, England). Sections were washed in PBS-T and incubated for a further 15 min in avidin–biotin-complex (Vector Laboratories, Peterborough, England). Sections were again washed in PBS-T for 5 min, followed by the addition of 3, 3′-diaminobenzidine (Sigma) for 7 min. Sections were immersed in tap water for 3 min to stop the chromogenic reaction and counterstained in Mayer's Haemalum (BDH, Poole, England). Finally, sections were dehydrated in alcohol, cleared in xylene and mounted in p-xylene-bis n-pyridinium bromide. Sections were allowed to dry for a minimum of 1 hr before viewing under a microscope (Olympus DP 50). An isotypic mouse IgG1 antibody (Dako CytoMation, Glostrup, Denmark) was used as a negative control.

Slides were scored independently by 2 investigators. The scoring method used for the semiquantification of LPB protein was based on both the intensity of the stain and the percentage of tumour cells stained. Intensity was scored as follows; negative = 0, low = 1, moderate = 2 and strong = 3. The percentage of tumour cells stained was scored as follows: negative = no staining, 1–10% cells staining = 1, 11–50% cells staining = 2 and 51–100% cells staining = 3. A total score was calculated by combining the intensity score and the percentage staining score.

Cell culture

The following cell lines used were; MDA-MB-231, MDA-MB-415, MCF-7, SK-BR-3, BT-474 and ZR-75-1 (American Type Culture Collection). The SKBR3, BT474 and ZR-75-1 cell were cultured in RPMI-1640, the MCF-7 cells in Eagle's MEM and the MDA-MB-231 cells and MDA-MB-415 cells in Leibovitz L-15. All culture media was supplemented with 10% (w/v) foetal calf serum, 2 mM L-glutamine, 20 mM Hepes, 50 unit/ml penicillin, 50 μg/ml streptomycin and 2 μg/ml fungizone (amphotericin B) (Gibco BRL, Gaithersburg, MD). Cells were maintained in humidified air with 5% CO₂ and extracted for RNA and protein as they approached confluence.

Other assays

ER and PR were measured by ELISA (Abbott Diagnostics, North Chicago, IL).15 The cut-off point for ER was 200 fmol/g wet weight tissue, while the cut-off for PR was 1,000 fmol/g wet weight tissue. Proliferation rates were measured in breast tissue extracts using an ELISA for proliferating cell nuclear antigen (PCNA) (Oncogene, CN Biosciences, Nottingham, England). MGA mRNA in the breast tissues was investigated by conventional PCR8 while mRNA in cell lines was investigated by real-time PCR, as previously described.10

Statistical analysis

The Spearman–Rank Correlation was used to compare continuous variables; the Mann–Whitney U test to compare categorical/nominal variables, and the Chi-Square test was used to compare purely categorical data. All statistics were calculated using StatView for Windows Version 5.0.1 (SAS Institute). A p-value < 0.05 was considered to be statistically significant.

Results

Expression of LPB mRNA in breast carcinomas, nonmalignant breast tissue and nonbreast tissue

Table III summarises the distribution of LPB mRNA in normal breast tissue, fibroadenomas, primary breast carcinomas and a miscellaneous panel of nonbreast tissues. For each type of breast tissue investigated, approximately 65–75% of the samples were found to express LPB mRNA. Median levels however, were approximately 4-fold higher in primary carcinomas compared to normal breast tissue (p = 0.02, Mann–Whitney U test).

Table III. Distribution of LPB mRNA Expression in Normal Breast Tissue, Fibroadenomas, Breast Carcinomas and Nonbreast Tissue

Tissue type	n	No expressing LPB1	Median	Range	95% CI
Levels were normalised against the housekeeping gene GAPDH. CI, confidence interval. 1 Values in parentheses are in percentages.
Normal breast	25	16 (64)	0.088	0–1.400	0.062–0.302
Fibroadenoma	30	23 (77)	0.273	0–1.680	0.204–0.404
Carcinoma	156	102 (65)	0.354	0–2.386	0.347–0.531
Nonbreast	36	8 (22)	0	0–0.768	0.01–0.09

In contrast to the different types of breast tissue, only 2/22 (9.1%) specimens of nonmalignant nonbreast tissues and 6/14 (42.8%) samples of malignant nonbreast tissues were found to have detectable LPB mRNA. The nonmalignant nonbreast tissues expressing LPB were specimens of skin and ovary whereas the malignant nonbreast samples expressing LPB were cancers of the colon, skin (basal cell carcinoma), ovary, parotid and two prostate cancers. As with the normal breast tissues, LPB mRNA levels were also significantly higher in the breast carcinomas compared to the nonbreast tissues (p < 0.001). As the median level of LPB was zero in the nonbreast tissues, it was not possible to calculate the fold-increase in the breast carcinomas based on this value.

Relationship between LPB expression and characteristics of the primary cancers

The relationship between LPB mRNA and tumor characteristics is summarized in Table IV. LPB mRNA levels were significantly higher in low-grade (i.e., Grades 1 and 2 combined) compared to high-grade tumors (Grade 3) (p < 0.0004, Mann–Whitney U test), in ER-positive compared to ER-negative cancers (p = 0.0448, Mann–Whitney U test) and in lobular compared to ductal cancer (p = 0.0188, Mann–Whitney U test). In contrast, no significant relationship was found between LPB mRNA levels and tumor size, lymph node status or PR-status. Of the 156 primary breast cancers investigated for LPB mRNA, 44 representative samples were available for measurement of cell proliferation rate using an ELISA for PCNA. For these 44 samples a weak but significant inverse relationship was found between LPB mRNA and PCNA levels (r = −0.28, p = 0.034, Spearman rank correlation test).

Table IV. Relationship Between the Frequency of LPB mRNA Expression in Primary Breast Carcinomas and Established Prognostic Indicators in Breast Cancer

Tumour characteristics	n	Positive (%)	p-value*	Median level	p-value§
ER, estrogen receptor; PR, progesterone receptor. * p-value according to the Chi-square test. § p-value according to the Mann–Whitney U test.
Size (cm)
≤2	40	65.0	0.6121	0.347	0.6121
>2	102	66.7		0.431
Unknown	14	57.1
Grade
1 and 2	52	82.7	0.0011	0.554	0.0004
3	75	54.7		0.202
Unknown	29	62.1
Nodes
Negative	65	66.2	0.7195	0.423	0.7985
Positive	75	69.3		0.422
Unknown	16	43.8
ER
Negative	58	58.6	0.0417	0.277	0.0448
Positive	83	75.9		0.476
Unknown	15	33.3
PR
Negative	92	66.3	0.5553	0.455	0.6530
Positive	44	72.7		0.347
Unknown	20	45.0
Age (years)
≤50	52	61.5	0.3142	0.334	0.8976
>50	77	68.8		0.433
Unknown	27	62.9
Histological type
Ductal	119	76.3	0.1012	0.311	0.0118
Lobular	20	75		0.700
Mixed ductal and lobular	5	100
Unknown	12	58.3

Relationship between LPB mRNA and MGA mRNA expression

Table V compares the expression of LPB and MGA mRNA in the different types of breast tissue as well as nonbreast tissue investigated. For each tissue type investigated, LPB mRNA was detected more frequently than MGA mRNA. Concordance in expression was found in 8/13 (62%) normal breast tissues, 13/15 (87%) fibroadenomas, 80/100 (80%) primary breast cancers and 6/10 (60%) nonbreast tissues. Discordance was found in 5/13 (38%) normal breast tissues, i.e., 5 expressed LPB in the absence of MGA, in 2/15 (13%) fibroadenomas, i.e., 2 expressed LPB in the absence of MGA, in 20/100 (20%) breast cancers, i.e., 16 (16%) expressed LPB in the absence of MGA and 4 (4%) expressed MGA in the absence of LPB and 4/10 (40%) nonbreast tissues, i.e., all 4 expressed LPB in the absence of MGA (Table VI).

Table V. Frequency of Expression of LPB mRNA and MGA mRNA in Breast and Nonbreast Tissues

Tissue type	n	No expressing LPB1	No expressing MGA1
1 Values in parentheses are in percentages.
Normal breast tissue	13	9 (75)	4 (33)
Fibroadenoma	15	10 (67)	8 (53)
Primary breast carcinoma	100	67 (67)	55 (55)
Non-breast tissue	10	4 (40)	0

Table VI. Expression Patterns of LPB mRNA and MGA mRNA in Breast and Non-Breast Tissues

Tissue type	Expression pattern of LPB and MGA1
	LPB+/MGA+	LPB+/MGA−	LPB−/MGA+	LPB−/MGA−
1 Values in parentheses are in percentages.
Normal breast tissue	4/13 (31)	5/13 (38)	0/13 (0)	4/13 (31)
Fibroadenoma	8/15 (54)	2/15 (13)	0/15 (0)	5/15 (33)
Primary breast carcinoma	51/100 (51)	16/100 (16)	4/100 (4)	29/100 (29)
Nonbreast tissue	0/10 (0)	4/10 (40)	0/10 (0)	6/10 (60)

Detection of LPB protein by Western blotting

Preliminary experiments were carried out to check the specificity of the LPB antibody. LPB mRNA expression was initially investigated in 6 different breast cancer cell lines, i.e., MDA-MB-231, MDA-MB-415, MCF-7, SK-BR-3, BT-474 and ZR-75-1 (American Type Culture Collection). Of these 6 cell lines, only 2, i.e., MDA-MB-415 and ZR-75-1 were found to express LPB mRNA. After Western blotting on the 6 cell lines, only the cells lines expressing LPB mRNA were found to contain a specific band migrating with the expected molecular mass of free LPB, i.e., 7–8 kDa (Table VII). These findings indicate that the antibody used is likely to be specific for free LPB.

Table VII. Expression of LPB mRNA and Protein in Different Breast Cancer Lines

Cell line	LPB mRNA (arbitrary units)	LPB protein (arbitrary units)
ND, not detectable. LPB mRNA levels were determined by real-time PCR while LPB protein was determined by Western blotting. For mRNA, LPB was related to GAPDH while for protein, LPB was related to beta-actin. Each value is mean of 2 experiments.
MDA-MB-231	ND	ND
MCF-7	ND	ND
BT-474	ND	ND
ZR-75-1	0.26	1.0
SK-BR-3	ND	ND
MDA-MB-415	0.69	1.12

Figure 1 shows a typical Western blot of LPB from breast carcinomas. As can be seen, samples 1, 3, 5 and 7 contained free LPB. One of the samples investigated (Sample 5, Fig. 1) however, contained additional bands. The identities of these bands are unknown but they may represent complex forms of LPB, e.g., LPB–MGA complexes.

Figure 1. Western blot of LPB protein from breast cancers. ZR: serum free medium from the ZR-75-1 breast cancer cell line was used as a positive control. Lanes 1–7 were extracts of 7 breast carcinomas. NEG, negative tissue control, i.e., an extract of normal colon.

Detection of LPB protein by immunohistochemistry

Immunohistochemistry of LPB in sections of invasive breast cancer showed that the peptide was predominantly expressed in the cytoplasm of epithelial cells (Fig. 2). Occasional staining was however found in stromal cells. This stromal cell staining was only evident when >50% of the tumor cells stained for LPB. Table VIII summarises the distribution of LPB staining scores in the 22 cancers investigated.

Figure 2. Immunohistochemical staining for LPB protein in paraffin embedded formalin-fixed breast cancer using the H85C21 monoclonal antibody (0.25 μg/ml). (a) negative isotypic mouse IgG1 control. (b) LPB positive staining in tumour cells. ×20 magnification was used.

Table VIII. Overall Staining Scores for LPB in 22 Sections from Formalin-Fixed Paraffin Embedded Invasive Breast Cancers

Overall score	No.	%
Overall score assigned to the 22 primary breast carcinoma sections used in our study. The overall score was calculated by combining the intensity score and the percent of positive cell staining score.
0	2	9.1%
1	0	0
2	1	4.5%
3	1	4.5%
4	6	27.3%
5	5	22.8%
6	7	31.8%

Discussion

Our results reported here show that the expression of LPB is preferentially but not exclusively confined to breast tissue. Our findings thus confirm and extend previous preliminary reports on the distribution of LPB in human tissue.7, 8, 9, 10, 12, 13 Compared to MGA however, expression of LPB was less specific for breast tissue. Other investigators have also found LPB to be less specific than MGA for breast tissue.7, 8, 9, 12, 13 Thus, Carter et al.9 detected LPB mRNA in the absence of MGA expression in a range of nonbreast tissues, including, skin and skeletal muscle. In our current study, LPB mRNA expression was found in 1/3 skin specimens. Lack of availability however, prevented us from investigating LPB in skeletal muscle.

Using substracted cDNA libaries and cDNA microarray, Jiang et al.7 previously reported that LPB was one of the most upregulated genes in breast cancer compared with nonbreast tissue. Consistent with these findings we show here using conventional RT-PCR that LPB mRNA levels in breast cancers were significantly increased relative to both normal breast tissue and nonbreast tissues. In contrast however, Zafrakas et al.13 using dot blot analysis on commercially available cDNA samples recently reported that LPB mRNA was downregulated in most breast cancers. The pathological and biochemical characteristics of the tumors investigated were not however, described in this report.13 As is clear from our results, expression levels of LPB in breast cancer depend on tumor grade, histology type and ER status.

As mentioned earlier, LPB in breast tissue is thought to exist as a complex with MGA.12 Consistent with the presence of such a complex, in general, we found good concordance between expression of LPB and MGA in the different types of breast tissue investigated. However, as 16/100 (16%) of breast cancers had measurable LPB mRNA in the absence of detectable MGA mRNA and 4/100 (4%) expressed MGA in the absence of measurable LPB, LPB may not always exist in complex with MGA in breast cancer. In addition, LPB was detected in a number of nonbreast tissues in the absence of detectable MGA. Similar to our findings, Zafrakas et al.13 also found expression of LPB in the absence of MGA. In some tissues, LPB may therefore exist either as a free form or in a complex with other secretoglobin proteins.

Our finding of a significant positive relationship between LPB expression and both low-grade and positive ER-status, combined with an inverse relationship between LPB and PCNA suggests that the presence of LPB in breast tumors is associated with good prognosis. Indeed, previously, high levels of MGA were found to predict good outcome in patients with breast cancer.16 The predictive impact of MGA was particularly strong in patients treated with adjuvant tamoxifen therapy.16 In future work, we therefore intend relating LPB levels to patient outcome.

To our knowledge, LPB has not so far been detected in blood. However, MGA protein has been found in serum from breast cancer patients17 while MGA mRNA has been detected in circulating tumor cells from such patients.18 Although LPB has not been detected in blood, antibodies against this peptide are known to be present in patients with advanced breast cancer.19 Assuming LPB protein can be released into the circulation, LPB is also a potential marker for aiding the detection and monitoring treatment in patients with breast cancer. A goal for future research should therefore be the development of an assay for detecting LPB in serum.