- Email: [email protected]
Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast
originals
Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast A. RUIBAL,a A. SÁNCHEZ SALMÓN,a M. GARRIDO,a A. BOGDAN CIOBOTARUa AND J.I. ARIASb a
Department of Nuclear Medicine. Hospital Clínico Universitario. School of Medicine. Santiago de Compostela. Spain. General Surgery Service. Hospital Monte del Naranco. Oviedo. Spain.
b
Abstract.—Objective. To study the possible correlations between the preoperative CA15.3 serum levels and the cellular proliferation, measured by S-phase (SP), in patients having infiltrating ductal carcinomas (IDC) of the breast Material and methods. The study group included 79 patients with an age ranged between 39 and 86 yrs (64.8 ± 11.8). Ploidy and S-phase were measured by cytometry (Fascam. Beckton Dikinson. USA) in fresh simples Results: Using as cut-off for SP the value of 7 %, which represents the median obtained previously in 321 patients with IDC (r: 0.8-51.2; 9.3 ± 7.9; percentiles 25 y 75; 4.3 y 11.8 %), we can observed that the antigenic levels were higher (p: 0.015) in the tumors with low SP. These same behavior was noted when 30U/ml was used as cut-off for CA15.3. Likewise, the levels of the tumor marker increased significantly (p: 0.007) when the SP moved from < 4.3 % to 7.1 %, to decrease later (p: 0.010) when the SP value was comprised between 7.11% and 11.8 %. The same behavior of this tumor marker in relation to the SP was noted in tumors without axillary involvement tumors, as well as in aneuploid carcinomas. Conclusion: a) Release of CA15.3 happens when SP increases to rise the 7.1 % value, to decrease later although that goes on increasing, and b) The same behaviour of this marker with the S-phase was observed in tumors without axillary involvement, as well as in aneuploid carcinomas.
11,8). La ploidía y FS fueron determinadas por citometría de flujo en muestras en fresco (Fascam. Beckton Dikinson. EE. UU.). Resultados. Tomando como dintel de positividad para la FS el valor de 7 %, que representa la mediana obtenida previamente en un grupo de 321 CDI (i: 0,8-51,2; 9,3 ± 7,9; percentiles 25 y 75; 4,3 y 11,8 %), observamos que las concentraciones del marcador fueron mayores (p: 0,015) en los casos con menor proliferación celular. Esto mismo se constató al valorar cualitativamente (> 30 U/ml) el marcador. Asimismo, las concentraciones de CA15.3 aumentaron significativamente (p = 0,007) al pasar la FS de < 4,3 % a FS comprendida entre 4,3 y 7,1 %, para luego descender (p = 0,010) en los casos con FS entre 7,11 y 11,8 % y no modificarse cuando aquella fue > 11,8 %. Este mismo comportamiento lo observamos en los tumores sin afectación axilar. Conclusiones. a) La liberación del CA15.3 ocurre cuando la FS se incrementa hasta alcanzar el valor del 7,1 %, para luego ir disminuyendo aunque aquella aumente, y b) este mismo comportamiento del marcador con la fase S se constató cuantitativamente en los tumores aneuploides y sin afectación axilar. PALABRAS CLAVE: CA15.3 sérico, proliferación celular, fase S, carcinoma ductal infiltrante, mama.
KEY WORDS: serum CA15.3, cellular proliferation, S-phase, infiltrating ductal carcinoma, breast.
INTRODUCTION CA15.3 SÉRICO Y PROLIFERACIÓN CELULAR EN CARCINOMAS DUCTALES INFILTRANTES DE MAMA Resumen.—Objetivo. Estudiar la posible correlación entre las concentraciones séricas preoperatorios de CA15.3 y la proliferación celular medida por la fase de síntesis (FS) en carcinomas ductales infiltrantes de mama (CDI). Material y métodos. El grupo estudio incluyó 79 pacientes de edades comprendidas entre los 39 y 86 años (64,8 ±
Received: 29-05-2007. Accepted: 19-07-2007. Correspondence: A. RUIBAL. Servicio de Medicina Nuclear. Hospital Clínico Universitario. 15706 Santiago de Compostela. Spain. E-mail: [email protected]
CA15.3 (MUC1) is a tumour marker that has been used widely for years in daily clinical practice.1 While its main application lies in the follow-up of breast cancer,2 serum elevations may be observed in other tumours,3 non-malignant situations4 and following pharmacological intervention5. Similarly, it may not only be detected in blood, but also in other biological fluids, including breast cysts.6 In breast tumours, its preoperative serum concentrations are related to tumour size, axillary nodular involvement, distance metastases and histological degree7-9 and behave, furthermore, following mutivariant analysis, as an independent prognosis factor of disease-free interval, both in the group considered globally and in positive axillary tumours.10 Its relationship with hormone dependence
Rev Esp Med Nucl. 2007;26(6):367-71
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Ruibal A et al. Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast
is not unanimous. Serum CA15.3 is also an indicator of a poorer response to chemotherapy, and together with lymphovascular invasion and the expression of the erbB2 encogen, it behaves as a prognosis factor in locally advanced breast cancer.11 MUC1, human polymorphic epithelial mucin, is a highly glycosylated transmembrane protein with protective functions that is aberrantly over-expressed in many tumours, including a high percentage of breast origin tumours. This is why it is regarded as a tumourassociated antigen. It behaves as an oncoprotein, and its greater cellular expression is correlated to tumour the aggressiveness and to lower survival. It also seems to play an important role in in situ ductal hyperplasiacarcinoma transition.12 At biological level, many of its actions have been seen to be carried out by means of biochemical mechanisms related to its cytoplasmic dimension. Thus, it intervenes in cell proliferation, apoptosis, invasion and transcription, and is directly correlated to the genes of the tumour necrosis factor (TNF), kinase serine/threonine RAF1 and the matrix metalloproteinase 2 (MMP2). At protein level it is related to the c-myc gene and phosphorylated AKt.13,14 Its C-terminal fragment interacts with the alpha estrogen receptor, and this is stimulated by estradiol, which induces the growth and survival of the tumour cells.15 Similarly, it inhibits E cadherin-mediated cell aggregation and the cytotoxic action of the K cells.16-18 Some variants modulate the localisation of certain growth factor receptors, which entails a different response to external stimuli19. Continuing in our line of work,8 and in view of the above, we have analysed, in women with infiltrating ductal carcinoma of the breast, the behaviour of serum CA15.3 according to the cell proliferation of the primitive tumour, measured by the cell synthesis phase (SP).
MATERIAL AND METHODS
The study group included 79 treatment-naive patients with infiltrating ductal carcinoma of the breast aged between 39 and 86 years (64.8 ± 11.8; median 66), in whom a preoperative serum determination of CA15.3 was performed. This was determined by means of a double clonal immunoradiometric method of CIS. BioInternational (France). Ploidy and sph were determined by flow cytometry in fresh samples (Fascam. Beckton Dikinson. USA). The tissue was 368
crushed with scissors to obtain a cellular dispersion and then filtered through a 100A pore mesh to separate the cores from the tumour stroma. The cores were resuspended in buffer citrate and frozen to –70 °C until determination by the Hedley technique. The DNA staining was performed with propidium iodide with fluorescein which binds stoichiometrically to the nucleic acids. To avoid binding to RNA, RNAase was added to the sample, as well as a proteolytic enzyme to break down the cellular membranes and the fibres of the stroma. The reading was verified on a flow cytometer (Becton Dickinson, USA.) and a programme (Cellfit) with several models, using RFITT and POLY, in our case. The quantity of fluorescein given off by the propidium iodide per cell is proportional to the amount of DNA. It was regarded as a diploid pattern when the DNA content was the same as the normal control (lymphocytes) and as an aneuploid pattern when it was different to the former. The percentage of cells was also obtained in the different phases of the cell cycle, with our study using the synthesis or S phase. More technical and analytical details have been expounded in other studies.20 Since the results obtained did not follow a normal distribution, we used nonparametric statistical (Mann Whitney) and Chi squares tests, and the Yates correction as necessary for the comparison of proportions. A difference was regarded as statistical when the p value was lower than 0.05.
RESULTS
As can be observed in table 1 the concentrations of serum CA15.3 did not differ statistically between diploid and aneuploid tumours. When we analysed the influence of the cell synthesis phase, taking the 7% that represents the median previously obtained in a group of 321 infiltrating ductal carcinomas as the positivity threshold (i: 0.8-51.2; 9.3 ± 7.9; percentiles 25 and 75; 4.3% and 11.8%), we observed that the concentrations of serum CA15.3 were greater (p = 0.015) in cases with less cell proliferation. On considering the positivity threshold of 30 U/ml for serum CA15.3, we observed the same behaviour (table 2). In an attempt to go into greater depth into the synthesis phase, we analysed the serum concentrations of CA15.3 according to the values of the former. As table 3 shows, tumour marker concentrations increased significantly (p = 0.007) in tumours with CSP between 4.3% and 7.1% versus those of SP < 4.3%, and then
Rev Esp Med Nucl. 2007;26(6):367-71
Ruibal A et al. Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast
fell (p = 0.010) in cases with SP between 7.11 and 11.8% and did not differ when the synthesis phase was > 11.8%. This same behaviour was quantitatively ascertained in aneuploid tumours, and qualitatively and quantitatively in cases without axillary nodular involvement (table 4).
DISCUSSION
In recent years we have begun to know some of the biological functions of many serum tumour markers broadly used in daily clinical practice. One of them is CA15.3, which defines the MUC1 mucin, transmembrane glycoprotein (50% carbohydrates), located on the apical cell surface, which it protects and which is abnormally expressed in many tumours. MUC1 intervenes in different biological functions and is related, in certain breast cancer cell lines, to the epidermal growth factor receptor (EGFR), so that receptor activation phosphorylates the cytoplasmic dimension of the mucin and promotes binding to betacatenin. Other factors that intervene in the regulation of MUC1 are the C delta protein kinase and interleukin 7. MUC1 has been seen to act as an oncogene, favouring tumour transformation, and it is known to be involved in the regulation of transcription through NF-kappa B factor, as well as in the blockade of betacatenin.21 In transformed cells, MUC1 prevents the differentiation and function of dendritic cells and favours immune system inefficacy. Its aberrant intracellular location in tumour cells is correlated to a poorer prognosis and a greater tumour aggressiveness. The overexpression of MUC1 proteins is associated with greater metastatic behaviour, the consequences of the genetic pattern modulated by certain polymorphisms.22 In vitro studies have also demonstrated that estradiol
Table 1 DISTRIBUTION OF THE PREOPERATIVE SERUM CONCENTRATIONS OF CA15.3 (U/ML) IN INFILTRATING DUCTAL CARCINOMAS OF THE BREAST CLASSIFIED ACCORDING TO PLOIDY AND CELL SYNTHESIS PHASE Parameter
No.
Interval
Median
p
SP > 7 SP = 7 Diploid Aneuploid
38 41 33 46
6.3-161 10-106 6.3-10 8.34-161
20.0 23 23 20
0.015 ns
SP: cell synthesis phase; ns: not significant.
Table 2 CONCENTRATIONS OF PREOPERATIVE SERUM CA15.3 > 30 U/ML IN INFILTRATING DUCTAL CARCINOMAS OF THE BREAST CLASSIFIED ACCORDING TO PLOIDY AND S PHASE Parameter
> 30
p.
SP >7 SP ≤ 7 Diploid Aneuploid
5/38 17/41 97/33 10/46
0.005 ns
SP: cell synthesis phase; ns: not significant.
duplicates the expression of MUC1 in the MCF7 hormone-dependent breast cancer line, as well as its passage into the culture medium in the same proportion, whereas tamoxifen had little inhibitory effect. In hormone-independent lines, PMA (phorbol-12myristate-13-acetate) also stimulates the synthesis and release of MUC1, and this effect is abolished by specific inhibitors of protein kinase C.22,23 Our study found no differences in the serum values of CA15.3 when ploidy (diploid versus aneuploid)
Table 3 DISTRIBUTION OF THE PREOPERATIVE SERUM CONCENTRATIONS OF CA15.3 (U/ML) IN INFILTRATING DUCTAL CARCINOMAS OF THE BREAST CLASSIFIED ACCORDING TO CELL SYNTHESIS PHASE VALUES S Phase
No.
Interval
Mean ± SD
Median
> 30 (U/ml)
> 40 (U/ml)
< 4.3%
21
21.0 ± 7.0
20
7.11-11.8
13
> 11.8%
23
22 p < 0.001 31.5 p = 0.009 25 ns 20
1 p = 0.005 11 p = 0.074 1 ns 2
0
4.3-7.1%
10-37 p = 0.007 10-106 p = 0.010 9-54.8 ns 6.3-161
38.5 ± 28.2 21.6 ± 11.7 25.3 ± 20
7 1 2
S Phase: cell synthesis phase; SD: standard deviation; ns: not significant.
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Ruibal A et al. Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast
Table 4 CONCENTRATIONS OF SERUM CA15.3 AND POSITIVITY PERCENTAGES (THRESHOLD 30 U/ML) IN INFILTRATING DUCTAL CARCINOMAS OF THE BREAST CLASSIFIED ACCORDING TO PLOIDY, CELL SYNTHESIS PHASE AND AXILLARY NODULAR INVOLVEMENT Parameters
> 30 U/ml
D + SP > 7%
1/8
p.
Interval
Median
6.3-54.8
23.5
10-106 8.3-161
23.0 17.0
10-98 6.3-42
24.5 20.0
10-45 11-161
25.0 19.0
10-106
23.0
ns D + SP = 7% A + SP > 7%
5/23 4/28
A + SP = 7% N – /SP > 7%
6/16 2/20
N – /SP = 7% N + /SP > 7%
7/21 3/18
N + /SP = 7%
5/20
p ns
ns
0.023
ns
0.022
ns
ns
D: diploid; A: aneuploid; N–: without axillary nodular involvement; N + : with axillary nodular involvement; SP: cell synthesis phase.
was considered, although there were differences with cell proliferation measured through the SP. Contrary to expectations, it was the less proliferative tumours (SP < 7%) that showed greatest concentrations of the marker, and the same occurred when we performed a qualitative assessment, taking 30 U/ml as the threshold. This finding led us to study the concentrations of CA15.3 in tumours classified according to the S phase, taking the median (7%) and the percentiles 25 (4.3%) and 75 (11.8%) previously obtained in a group of 321 infiltrating ductal carcinomas of the breast as thresholds. We saw that the concentrations of CA15.3 increased significantly when the S phase went from < 4.3% to that which is comprised between 4.3% and 7.1% and then dropped when it was between 7.1% and 11.8% and was maintained in cases > 11.8%. This same pattern of behaviour was seen on considering the positivity of the marker (threshold 30 U/ml). It would therefore seem that the synthesis and release of the marker takes place when cell proliferation increases moderately and then falls as the latter advances. Another possibility is that CA15.3 was synthesised, but not released, from the cell surface. These facts do not contradict the observations of other authors in colorectal cancer24, and in human fibroblasts, where Oshimo S et al25 observed that KL-6, a MUC1 mucin, promoted proliferation and inhibited apoptosis, which is of great physiopathological interest. This behaviour of serum CA15.3 differed to that of 370
cytokeratin 19 (cyfra 21.1), linked to apoptosis like other keratins.26 We have already said that there were no differences in the serum concentrations of CA15.3 between diploid and aneuploid tumours. When we classified the IDC according to the ploidy and S phase, we saw that, quantitatively, the aneuploid tumours with low S phase (< 7%) coursed significantly with higher concentrations of antigen. On considering axillary nodular involvement, we saw that in the absence of the latter, tumours with A phase < 7% course qualitatively and quantitatively with greater concentrations of the marker, whereas no differences were observed in the presence of axillary involvement, which suggests that elevations of the antigen are linked to regional dissemination, but not to cell proliferation. This behaviour of CA15.3 was not observed with the carcinoembryonic antigen (CEA) (data not shown). Our results lead us to the following considerations: 1. The release of CA15.3 occurs when percentages of SP are increased until the value of 7.1% is reached, and is then reduced, although proliferation continues to increase. 2. This same behaviour of the marker according to the S phase was ascertained in the aneuploid tumours and without axillary nodular involvement.
BIBLIOGRAPHY 1. Ruibal A. El CA15.3, marcador del cáncer de mama. Med Clin (Barc). 1988;91:341-3. 2. Colomer R, Ruibal A, Genollá J, Rubio D, del Campo JM, Boda R, et al. Circulating CA15.3 levels in the postsurgical follow-up of breast cancer patients and in non malignant diseases. Breast Cancer Res Treat. 1989;13:123-33. 3. Colomer R, Ruibal A, Genolla J, Salvador L. Circulating CA15.3 antigen levels in non-mammary malignancies. Br J Cancer. 1989;59:283-6. 4. Ruibal A, Encabo G, Genollá J, Guarga A, Urrutia A, Colomer R. Serum CA15.3 levels in patients with general pathology and malignant diseases (excluding breast cancer). Bull Cancer. 1986;73:94-5. 5. Pentheroudakis G, Malamou-Mitsi V, Briasoulis E, Damala K, Vassou A, Vartholomatos G, et al. The neutrophil, not the tumor: serum CA15-3 elevation as a result of granulocyte-colony stimulating factor induced neutrophil MU C1 overexpression and neutrophilia in patients with breast carcinoma receiving adjuvant chemotherapy. Cancer. 2004;101:1767-75. 6. Vizoso F, Allende MT, Fueyo A, Vigal G, García Moran M, Ruibal A. CA15.3 behavior in cystic breast disease. Int J Biol Markers. 1989;4:181-2. 7. Lumachi F, Basso SM, Brandes AA, Pagano D, Ermani M. Relationship between tumor markers CEA and CA15.3, TNM staging, estrogen receptor rate and MIB1 index in patients with pT1-2 breast cancer. Anticancer Res. 2004;24:3221-4.
Rev Esp Med Nucl. 2007;26(6):367-71
Ruibal A et al. Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast
8. Ruibal A, Garrido M, Arias JI. Concentraciones séricas preoperatorios de CA15.3 y CEA y su correlación con los parámetros clínico-biológicos del cáncer de mama. Rev Esp Med Nucl. 2006;25:180-3. 9. Molina R, Jo J, Filella X, Zanón G, Pahisa J, Nuñoz M, et al. cerbB-2 oncoprotein, CEA, and CA 15.3 in patients with breast cancer: prognostic value. Breast Cancer Res Treat. 1998;51: 109-19. 10. Martín A, Corte MD, Álvarez AM, Rodríguez JC, Andicoechea A, Bongera M, et al. Prognostric value of pré-operative serum CA15.3 levels in breast câncer. Anticancer Res. 2006;26:3965-71. 11. Al-Azawi D, Kelly G, MMyers E, McDermott EW, Hill AD, Duffy MJ, et al. CA15.3 is predictive of response and disease recurrente following treatment in locally advanced breast cancer. BMV Cancer. 2006;6:220. 12. Moomers EC, Leonhart AM, von Mensdorff-Pouilly S, Schol DJ, Hilgers J, Meijer CJ, et al. Aberrant expression of MUC1 mucon in ductal hyperplasia, and ductal carcinoma in situ of the breast. Int J Cancer. 1999;84:466-9. 13. Hattrup CL, Gendler SJ. MUC1 Alters oncogenic events and transcription in human breast cancer cells. Breast Cancer Res. 2006;8:R37. 14. Tsutsumida H, Swanson BJ, Singh PK, Caffrey TC, Kitajima S, Goto M, et al. RNA interference suppression of MUC1 reduces the growth rate and metastatic phenotype of human pancreatic cancer cells. Clin Cancer Res. 2006;12:2976-87. 15. Wei X, Xu H, Kufe D. MUC1 oncoprotein stabilizes and activates estrogen receptor alpha. Mol Cell. 2006;21:295-305. 16. Wwsseling J, van der Valk SW, Vos HL, Sonnenberg A, Hilkens J. Episialin (MUC1) overexpression inhibits integrin-mediated cell adhession to extracellular matriz components. J Cell Biol. 1995;129:255-65. 17. Heuser C, Ganser M, Hombach A, Brand H, Denton G, Hanish FG, et al. An anti-MUC1 antibody –interleukin 2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumor cells. Br J Cancer. 2003;89:1130-9.
18. Doi M, Yokoyama A, Kondo K, Ohnishi H, Ishikawa N, Hattori N, et al. Anti-tumor effect of the anti-KL-6/MUC1 monoclonal antibody through exposure of surface molecules by MUC1 capoing. Cancer Sci. 2006;97:420-9. 19. Carraway KL 3rd, Funes M, Workman HC, Sweeney C. Contribution of membrana mucins to tumor progression through modulation of cellular growth signaling pathways. Curr Top Dev Biol. 2007;78:1-22. 20. Ruibal A, Arias JI, del Río MC, Lapeña G, Schneider J, Tejerina A. Histological grade in breast cancer: association with clinical and biological features in a series of 229 patients. Int J Biol Markers. 2001;16:56-61. 21. Thompson EJ, Shanmugam K, Hattrup CL, Kotlarczyk KL, Gutiérrez A, Bradley JM, et al. Tyrosines in the MUC1 cyttoplasmatic tail modulate transcription via the extracellular signal-tegulated kinase 1/2 and nuclear factor-kappa B pathways. Mol Cancer Res. 2006;4:489-97. 22. Schmid BC, Buluwela L, Liu Q, Fasching B, Tong D, Stimpfl M, et al. Expression of MUC1 splice variants correlates with invasive growth of breast câncer cell lines. Breast Cancer Res Treat. 2002;76:211-9. 23. Porowska H, Paszkiewicz-Gadek A, Wolczynski S, Gindzienski A. MUC1 expression in human breast cancer cells is alterated by the factors affecting cell proliferation. Neoplasma. 2002;49: 104-9. 24. Kimura T, Tanaka S, Haruma K, Sumii K, Kajiyama G, Shimamoto F, et al. Clinical significance of MUC1 and E-cadherin expression, cellular proliferation and angiogenersis at the deepest invasive portion of colorectal cancer. Int J Oncol. 2000;16:55-64. 25. Ohshimo S, Yokoyama A, Hattori N, Ishikawa N, Hirasawa Y, Kohno N. KL-6, a human MUC1 mucin, promotes proliferation and survival of lung fibroblasts. Biochem Biophys Res Commun. 2005;338:1845-52. 26. Barak V, Goike H, Panaretakis KW, Einarsson R. Clinical utility of cytokeratins as tumor markers. Clin Biochem. 2004;37:529-40.
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Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast A. RUIBAL,a A. SÁNCHEZ SALMÓN,a M. GARRIDO,a A. BOGDAN CIOBOTARUa AND J.I. ARIASb a
Department of Nuclear Medicine. Hospital Clínico Universitario. School of Medicine. Santiago de Compostela. Spain. General Surgery Service. Hospital Monte del Naranco. Oviedo. Spain.
b
Abstract.—Objective. To study the possible correlations between the preoperative CA15.3 serum levels and the cellular proliferation, measured by S-phase (SP), in patients having infiltrating ductal carcinomas (IDC) of the breast Material and methods. The study group included 79 patients with an age ranged between 39 and 86 yrs (64.8 ± 11.8). Ploidy and S-phase were measured by cytometry (Fascam. Beckton Dikinson. USA) in fresh simples Results: Using as cut-off for SP the value of 7 %, which represents the median obtained previously in 321 patients with IDC (r: 0.8-51.2; 9.3 ± 7.9; percentiles 25 y 75; 4.3 y 11.8 %), we can observed that the antigenic levels were higher (p: 0.015) in the tumors with low SP. These same behavior was noted when 30U/ml was used as cut-off for CA15.3. Likewise, the levels of the tumor marker increased significantly (p: 0.007) when the SP moved from < 4.3 % to 7.1 %, to decrease later (p: 0.010) when the SP value was comprised between 7.11% and 11.8 %. The same behavior of this tumor marker in relation to the SP was noted in tumors without axillary involvement tumors, as well as in aneuploid carcinomas. Conclusion: a) Release of CA15.3 happens when SP increases to rise the 7.1 % value, to decrease later although that goes on increasing, and b) The same behaviour of this marker with the S-phase was observed in tumors without axillary involvement, as well as in aneuploid carcinomas.
11,8). La ploidía y FS fueron determinadas por citometría de flujo en muestras en fresco (Fascam. Beckton Dikinson. EE. UU.). Resultados. Tomando como dintel de positividad para la FS el valor de 7 %, que representa la mediana obtenida previamente en un grupo de 321 CDI (i: 0,8-51,2; 9,3 ± 7,9; percentiles 25 y 75; 4,3 y 11,8 %), observamos que las concentraciones del marcador fueron mayores (p: 0,015) en los casos con menor proliferación celular. Esto mismo se constató al valorar cualitativamente (> 30 U/ml) el marcador. Asimismo, las concentraciones de CA15.3 aumentaron significativamente (p = 0,007) al pasar la FS de < 4,3 % a FS comprendida entre 4,3 y 7,1 %, para luego descender (p = 0,010) en los casos con FS entre 7,11 y 11,8 % y no modificarse cuando aquella fue > 11,8 %. Este mismo comportamiento lo observamos en los tumores sin afectación axilar. Conclusiones. a) La liberación del CA15.3 ocurre cuando la FS se incrementa hasta alcanzar el valor del 7,1 %, para luego ir disminuyendo aunque aquella aumente, y b) este mismo comportamiento del marcador con la fase S se constató cuantitativamente en los tumores aneuploides y sin afectación axilar. PALABRAS CLAVE: CA15.3 sérico, proliferación celular, fase S, carcinoma ductal infiltrante, mama.
KEY WORDS: serum CA15.3, cellular proliferation, S-phase, infiltrating ductal carcinoma, breast.
INTRODUCTION CA15.3 SÉRICO Y PROLIFERACIÓN CELULAR EN CARCINOMAS DUCTALES INFILTRANTES DE MAMA Resumen.—Objetivo. Estudiar la posible correlación entre las concentraciones séricas preoperatorios de CA15.3 y la proliferación celular medida por la fase de síntesis (FS) en carcinomas ductales infiltrantes de mama (CDI). Material y métodos. El grupo estudio incluyó 79 pacientes de edades comprendidas entre los 39 y 86 años (64,8 ±
Received: 29-05-2007. Accepted: 19-07-2007. Correspondence: A. RUIBAL. Servicio de Medicina Nuclear. Hospital Clínico Universitario. 15706 Santiago de Compostela. Spain. E-mail: [email protected]
CA15.3 (MUC1) is a tumour marker that has been used widely for years in daily clinical practice.1 While its main application lies in the follow-up of breast cancer,2 serum elevations may be observed in other tumours,3 non-malignant situations4 and following pharmacological intervention5. Similarly, it may not only be detected in blood, but also in other biological fluids, including breast cysts.6 In breast tumours, its preoperative serum concentrations are related to tumour size, axillary nodular involvement, distance metastases and histological degree7-9 and behave, furthermore, following mutivariant analysis, as an independent prognosis factor of disease-free interval, both in the group considered globally and in positive axillary tumours.10 Its relationship with hormone dependence
Rev Esp Med Nucl. 2007;26(6):367-71
367
Ruibal A et al. Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast
is not unanimous. Serum CA15.3 is also an indicator of a poorer response to chemotherapy, and together with lymphovascular invasion and the expression of the erbB2 encogen, it behaves as a prognosis factor in locally advanced breast cancer.11 MUC1, human polymorphic epithelial mucin, is a highly glycosylated transmembrane protein with protective functions that is aberrantly over-expressed in many tumours, including a high percentage of breast origin tumours. This is why it is regarded as a tumourassociated antigen. It behaves as an oncoprotein, and its greater cellular expression is correlated to tumour the aggressiveness and to lower survival. It also seems to play an important role in in situ ductal hyperplasiacarcinoma transition.12 At biological level, many of its actions have been seen to be carried out by means of biochemical mechanisms related to its cytoplasmic dimension. Thus, it intervenes in cell proliferation, apoptosis, invasion and transcription, and is directly correlated to the genes of the tumour necrosis factor (TNF), kinase serine/threonine RAF1 and the matrix metalloproteinase 2 (MMP2). At protein level it is related to the c-myc gene and phosphorylated AKt.13,14 Its C-terminal fragment interacts with the alpha estrogen receptor, and this is stimulated by estradiol, which induces the growth and survival of the tumour cells.15 Similarly, it inhibits E cadherin-mediated cell aggregation and the cytotoxic action of the K cells.16-18 Some variants modulate the localisation of certain growth factor receptors, which entails a different response to external stimuli19. Continuing in our line of work,8 and in view of the above, we have analysed, in women with infiltrating ductal carcinoma of the breast, the behaviour of serum CA15.3 according to the cell proliferation of the primitive tumour, measured by the cell synthesis phase (SP).
MATERIAL AND METHODS
The study group included 79 treatment-naive patients with infiltrating ductal carcinoma of the breast aged between 39 and 86 years (64.8 ± 11.8; median 66), in whom a preoperative serum determination of CA15.3 was performed. This was determined by means of a double clonal immunoradiometric method of CIS. BioInternational (France). Ploidy and sph were determined by flow cytometry in fresh samples (Fascam. Beckton Dikinson. USA). The tissue was 368
crushed with scissors to obtain a cellular dispersion and then filtered through a 100A pore mesh to separate the cores from the tumour stroma. The cores were resuspended in buffer citrate and frozen to –70 °C until determination by the Hedley technique. The DNA staining was performed with propidium iodide with fluorescein which binds stoichiometrically to the nucleic acids. To avoid binding to RNA, RNAase was added to the sample, as well as a proteolytic enzyme to break down the cellular membranes and the fibres of the stroma. The reading was verified on a flow cytometer (Becton Dickinson, USA.) and a programme (Cellfit) with several models, using RFITT and POLY, in our case. The quantity of fluorescein given off by the propidium iodide per cell is proportional to the amount of DNA. It was regarded as a diploid pattern when the DNA content was the same as the normal control (lymphocytes) and as an aneuploid pattern when it was different to the former. The percentage of cells was also obtained in the different phases of the cell cycle, with our study using the synthesis or S phase. More technical and analytical details have been expounded in other studies.20 Since the results obtained did not follow a normal distribution, we used nonparametric statistical (Mann Whitney) and Chi squares tests, and the Yates correction as necessary for the comparison of proportions. A difference was regarded as statistical when the p value was lower than 0.05.
RESULTS
As can be observed in table 1 the concentrations of serum CA15.3 did not differ statistically between diploid and aneuploid tumours. When we analysed the influence of the cell synthesis phase, taking the 7% that represents the median previously obtained in a group of 321 infiltrating ductal carcinomas as the positivity threshold (i: 0.8-51.2; 9.3 ± 7.9; percentiles 25 and 75; 4.3% and 11.8%), we observed that the concentrations of serum CA15.3 were greater (p = 0.015) in cases with less cell proliferation. On considering the positivity threshold of 30 U/ml for serum CA15.3, we observed the same behaviour (table 2). In an attempt to go into greater depth into the synthesis phase, we analysed the serum concentrations of CA15.3 according to the values of the former. As table 3 shows, tumour marker concentrations increased significantly (p = 0.007) in tumours with CSP between 4.3% and 7.1% versus those of SP < 4.3%, and then
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Ruibal A et al. Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast
fell (p = 0.010) in cases with SP between 7.11 and 11.8% and did not differ when the synthesis phase was > 11.8%. This same behaviour was quantitatively ascertained in aneuploid tumours, and qualitatively and quantitatively in cases without axillary nodular involvement (table 4).
DISCUSSION
In recent years we have begun to know some of the biological functions of many serum tumour markers broadly used in daily clinical practice. One of them is CA15.3, which defines the MUC1 mucin, transmembrane glycoprotein (50% carbohydrates), located on the apical cell surface, which it protects and which is abnormally expressed in many tumours. MUC1 intervenes in different biological functions and is related, in certain breast cancer cell lines, to the epidermal growth factor receptor (EGFR), so that receptor activation phosphorylates the cytoplasmic dimension of the mucin and promotes binding to betacatenin. Other factors that intervene in the regulation of MUC1 are the C delta protein kinase and interleukin 7. MUC1 has been seen to act as an oncogene, favouring tumour transformation, and it is known to be involved in the regulation of transcription through NF-kappa B factor, as well as in the blockade of betacatenin.21 In transformed cells, MUC1 prevents the differentiation and function of dendritic cells and favours immune system inefficacy. Its aberrant intracellular location in tumour cells is correlated to a poorer prognosis and a greater tumour aggressiveness. The overexpression of MUC1 proteins is associated with greater metastatic behaviour, the consequences of the genetic pattern modulated by certain polymorphisms.22 In vitro studies have also demonstrated that estradiol
Table 1 DISTRIBUTION OF THE PREOPERATIVE SERUM CONCENTRATIONS OF CA15.3 (U/ML) IN INFILTRATING DUCTAL CARCINOMAS OF THE BREAST CLASSIFIED ACCORDING TO PLOIDY AND CELL SYNTHESIS PHASE Parameter
No.
Interval
Median
p
SP > 7 SP = 7 Diploid Aneuploid
38 41 33 46
6.3-161 10-106 6.3-10 8.34-161
20.0 23 23 20
0.015 ns
SP: cell synthesis phase; ns: not significant.
Table 2 CONCENTRATIONS OF PREOPERATIVE SERUM CA15.3 > 30 U/ML IN INFILTRATING DUCTAL CARCINOMAS OF THE BREAST CLASSIFIED ACCORDING TO PLOIDY AND S PHASE Parameter
> 30
p.
SP >7 SP ≤ 7 Diploid Aneuploid
5/38 17/41 97/33 10/46
0.005 ns
SP: cell synthesis phase; ns: not significant.
duplicates the expression of MUC1 in the MCF7 hormone-dependent breast cancer line, as well as its passage into the culture medium in the same proportion, whereas tamoxifen had little inhibitory effect. In hormone-independent lines, PMA (phorbol-12myristate-13-acetate) also stimulates the synthesis and release of MUC1, and this effect is abolished by specific inhibitors of protein kinase C.22,23 Our study found no differences in the serum values of CA15.3 when ploidy (diploid versus aneuploid)
Table 3 DISTRIBUTION OF THE PREOPERATIVE SERUM CONCENTRATIONS OF CA15.3 (U/ML) IN INFILTRATING DUCTAL CARCINOMAS OF THE BREAST CLASSIFIED ACCORDING TO CELL SYNTHESIS PHASE VALUES S Phase
No.
Interval
Mean ± SD
Median
> 30 (U/ml)
> 40 (U/ml)
< 4.3%
21
21.0 ± 7.0
20
7.11-11.8
13
> 11.8%
23
22 p < 0.001 31.5 p = 0.009 25 ns 20
1 p = 0.005 11 p = 0.074 1 ns 2
0
4.3-7.1%
10-37 p = 0.007 10-106 p = 0.010 9-54.8 ns 6.3-161
38.5 ± 28.2 21.6 ± 11.7 25.3 ± 20
7 1 2
S Phase: cell synthesis phase; SD: standard deviation; ns: not significant.
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Ruibal A et al. Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast
Table 4 CONCENTRATIONS OF SERUM CA15.3 AND POSITIVITY PERCENTAGES (THRESHOLD 30 U/ML) IN INFILTRATING DUCTAL CARCINOMAS OF THE BREAST CLASSIFIED ACCORDING TO PLOIDY, CELL SYNTHESIS PHASE AND AXILLARY NODULAR INVOLVEMENT Parameters
> 30 U/ml
D + SP > 7%
1/8
p.
Interval
Median
6.3-54.8
23.5
10-106 8.3-161
23.0 17.0
10-98 6.3-42
24.5 20.0
10-45 11-161
25.0 19.0
10-106
23.0
ns D + SP = 7% A + SP > 7%
5/23 4/28
A + SP = 7% N – /SP > 7%
6/16 2/20
N – /SP = 7% N + /SP > 7%
7/21 3/18
N + /SP = 7%
5/20
p ns
ns
0.023
ns
0.022
ns
ns
D: diploid; A: aneuploid; N–: without axillary nodular involvement; N + : with axillary nodular involvement; SP: cell synthesis phase.
was considered, although there were differences with cell proliferation measured through the SP. Contrary to expectations, it was the less proliferative tumours (SP < 7%) that showed greatest concentrations of the marker, and the same occurred when we performed a qualitative assessment, taking 30 U/ml as the threshold. This finding led us to study the concentrations of CA15.3 in tumours classified according to the S phase, taking the median (7%) and the percentiles 25 (4.3%) and 75 (11.8%) previously obtained in a group of 321 infiltrating ductal carcinomas of the breast as thresholds. We saw that the concentrations of CA15.3 increased significantly when the S phase went from < 4.3% to that which is comprised between 4.3% and 7.1% and then dropped when it was between 7.1% and 11.8% and was maintained in cases > 11.8%. This same pattern of behaviour was seen on considering the positivity of the marker (threshold 30 U/ml). It would therefore seem that the synthesis and release of the marker takes place when cell proliferation increases moderately and then falls as the latter advances. Another possibility is that CA15.3 was synthesised, but not released, from the cell surface. These facts do not contradict the observations of other authors in colorectal cancer24, and in human fibroblasts, where Oshimo S et al25 observed that KL-6, a MUC1 mucin, promoted proliferation and inhibited apoptosis, which is of great physiopathological interest. This behaviour of serum CA15.3 differed to that of 370
cytokeratin 19 (cyfra 21.1), linked to apoptosis like other keratins.26 We have already said that there were no differences in the serum concentrations of CA15.3 between diploid and aneuploid tumours. When we classified the IDC according to the ploidy and S phase, we saw that, quantitatively, the aneuploid tumours with low S phase (< 7%) coursed significantly with higher concentrations of antigen. On considering axillary nodular involvement, we saw that in the absence of the latter, tumours with A phase < 7% course qualitatively and quantitatively with greater concentrations of the marker, whereas no differences were observed in the presence of axillary involvement, which suggests that elevations of the antigen are linked to regional dissemination, but not to cell proliferation. This behaviour of CA15.3 was not observed with the carcinoembryonic antigen (CEA) (data not shown). Our results lead us to the following considerations: 1. The release of CA15.3 occurs when percentages of SP are increased until the value of 7.1% is reached, and is then reduced, although proliferation continues to increase. 2. This same behaviour of the marker according to the S phase was ascertained in the aneuploid tumours and without axillary nodular involvement.
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