Dissertação apresentada ao Programa de Pós-Graduação em Patologia da Faculdade de Medicina de Botucatu, Universidade Estadual Paulista - UNESP, como parte dos requisitos para obtenção do título de Mestre.
FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉCNICA DE AQUISIÇÃO E TRATAMENTO DA INFORMAÇÃO
DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CAMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: Selma Maria de Jesus
Paiva, Carlos Eduardo.
Avaliação da expressão dos genes TGFB1 e TGFBR2 em amostras de carcinomas ductais invasivos / Carlos Eduardo Paiva. – Botucatu : [s.n.], 2008
Dissertação (mestrado) – Universidade Estadual Paulista, Faculdade de Medicina de Botucatu, 2008.
Orientadora: Silvia Regina Rogatto Assunto CAPES: 40105008
1. Mamas - Câncer - Aspectos genéticos CDD 616.994
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The TGFB1 as a prognostic marker in human invasive breast cancers
CE Paivaa*, SA Drigob, FE Rosab, FA Moraes Netoc, MAC Dominguesd, JRF Caldeirae,
SR Rogattob
a Oncological and Hemato-oncological Center, São Paulo State University, Botucatu, São Paulo, Brazil
b NeoGene Laboratory, Department of Urology, São Paulo State University, Botucatu, São Paulo, Brazil
c Department of Pathology, Amaral Carvalho Hospital, Jau, São Paulo, Brazil d Department of Pathology, São Paulo State University, Botucatu, São Paulo, Brazil e Department of Senology, Amaral Carvalho Hospital, Jau, São Paulo, Brazil
Running title: TGFB1 and TGFBR2 genes in breast cancer
* Corresponding author. Rua Antônio Nunes da Silva Sobrinho, 180, Jardim Paraíso II, Botucatu-SP-Brasil. CEP:18.610-170. Tel.: +55 14 3814 3640. E-mail address:
Abstract
Breast carcinoma (BC) is currently regarded as a complex and heterogeneous disease presenting abnormalities in several molecular pathways. Although BC is the leading female cancer in incidence and the second one in mortality worldwide, the prognostic and predictive markers known are incapable to predict the outcome of all patients as well as to avoid unnecessary treatments. Two potential tumor markers with possible clinical implications are TGFB1 and its receptor TGFBR2. However, the role of TGFB1 in carcinogenesis and in the BC progression is not totally understood. The present study was conducted to determine the expression pattern and predictive / prognostic value of TGFB1 and TGFBR2 in breast tumors. A series of 49 primary ductal invasive carcinomas and seven normal breast tissues were evaluated by quantitative real time PCR (qRT-PCR) and immunohistochemistry (IHC). It was detected that low levels of TGFB1 and TGFBR2 transcripts were significantly correlated with tumors presenting aggressive phenotypes (high proliferation index and advanced clinical staging). In addition, low expression levels of TGFB1 protein in tumor cells were associated with a lower disease-free survival. It was observed that the tumors presented highly diminished TGFBR2 transcript expression in comparison to normal samples. In overall, these results suggest that TGFB1 and TGFBR2 downexpression is an important event in breast carcinogenesis. TGFB1 protein expression levels can be useful in clinical practice as a prognostic marker in mammary carcinomas.
1. Introduction
Breast cancer (BC) is the most frequent female neoplasia and is the second leading cause of death by malignant disease among North American and Brazilian women [1,2]. BC is a group of heterogeneous neoplasms characterized by distinct morphological appearances, genetic alterations and biological behaviors. Over the last decades, a large number of alterations have been described and implicated in the development and progression of the breast cancer; however, the known prognostic factors are incapable to predict the progression of all patients. Currently, the only recommended prognostic and predictive markers in breast cancer are estrogen and progesterone receptors and ERBB2/HER-2 oncogene status [3]. Consequently, efforts have been done to discover biomarkers related to early detection, treatment response, and clinical outcome such as metastasis, recurrence, and survival. Understanding the molecular events and the development of reliable biomarkers will ultimately offer novel therapeutic strategies and to improve the clinical management of patients with breast cancer.
The proliferation of neoplasic breast epithelial cells is regulated by different stimuli including cytokines and growth factors [4], such as the transforming-growth factor
(TGF-). TGF- , a pluripotent cytokine, plays an important role in wound healing, angiogenesis, and immunoregulation [5]. It has been suggested that TGF- participates in normal mammary gland morphogenesis and growth [6]. Three TGF- isoforms are expressed in mammals (TGFB1, TGFB2 and TGFB3) and their effects are mediated by cell surface receptors named as TGFBR1, TGFBR2 and TGFBR3. TGFB1 is the most studied and universally expressed isoform. TGFB1 binds with high affinity to the TGFBR2, this phosphorilates TGFBR1 initiating the intracellular signaling cascade [7].
It has been proposed that TGFB1 acts as a tumor suppressor in early stages of neoplasia, by inhibiting epithelial cell proliferation and inducing apoptosis. Besides, it has been suggested that TGFB1 can also act as a tumor promoter in later stages of carcinogenesis, by inducing an invasive and pro-metastatic phenotype characterized by an epithelial-to-mesenchymal transition (EMT) [8,9,10].
risk of subsequent invasive breast cancer [11]. Recently, it was demonstrated that some members of the TGF- signaling pathway (including TGFBR2) are commonly down-regulated in breast cancer cell lines and in breast cancer tissues samples. Interestingly, TGFBR2 gene suppression was associated with chromatin repression rather than DNA methylation [12].
Studies addressing the prognostic role of TGFB1 in human BC have shown conflicting results. TGFB1 mRNA expression level detected by semi-quantitative method in BC samples was significantly higher in patients with a favorable outcome as compared to patients with a poor prognosis [13]. Other studies have shown that high expression of TGFB1 protein was related to worst progression of the disease [14,15]. More recently, Koumoundourou et al. showed that low expression of TGFB1 protein was related to recurrence in T1-2N0 breast tumors [16].
2. Materials and methods
2.1. Patients
Forty nine ductal breast carcinoma samples were obtained from 2000 to 2004 from Amaral Carvalho Hospital, Jaú (São Paulo, Brazil). The patients were accrued consecutively and had no previous histological diagnosis of BC. The patients had undergone segmental resection or mastectomy, and none of them had received radiotherapy or chemotherapy prior to surgery. Five histopathologically normal mammary tissues used as a control were obtained from patients that undergoing mammary reduction. All patients were advised of the procedures and provided written informed consent approved by the Institution Ethics Committee (CEPFHAC 020/06).
Patients were followed every four months post surgery and the mean follow-up was 52.3 ± 19.8 months (13 to 83 months). Immediately after surgery, the tumor samples were
frozen at -80ºC. Frozen tissue sections were histopathologically evaluated and microdissected to ensure the presence of at least 90% of tumor cells. Histopathological classification was performed according to the WHO International Classification of Disease for Oncology [17], and the clinical stage was determined according to the UICC TNM classification [18]. The malignancy of infiltrating carcinomas was scored according to the Scarff-Bloom and Richardson grading system [19].
The patients received different chemotherapy treatments: AC (adriamycin, cyclophosphamide); FEC fluorouracil, epirubicin, cyclophosphamide), FAC (fluorouracil, adriamycin, cyclophosphamide); CMF (cyclophosphamide, methotrexate, 5-fluorouracil). Thirty-six patients (73%) received radiotherapy. At the end of chemotherapy treatment, 32 patients (65%), that were estrogen receptor (ESR1) positive and/or progesterone receptor (PGR) positive cases according to immunohistochemistry (IHC) analysis were treated with tamoxifen (20 mg/day) for 60 months.
2.2. Total RNA isolation and reverse transcription
Technologies, Rockville, MD, U.S.A.) in 10X Dnase I Reaction Buffer and 25 mM EDTA pH 8.0. The reaction was carried out in a PTC-100 thermal cycle (Peltier - Effect Cycling - MJ Research, Inc., USA) for 15 min at room temperature and the enzyme was inactivated by heating at 70ºC for 10 min. RNA was reverse transcribed in a final volume of 20µl containing 5x First-Strand Buffer (Invitrogen Life Technologies, Inc., Carlsbad, CA), 10 mM each dNTP, 0.5µg/µl Oligo (dT)18, 0.1 M dTT and SuperScriptTM II reverse transcriptase 200 U (Invitrogen Life Technologies, Inc., Carlsbad, CA). Reverse transcription was carried out for 60 min at 42ºC and the reaction mixture was subsequently inactivated for 15 min at 70ºC. The cDNA was stored at -70ºC.
2.3. Quantitative RT-PCR
The PCR amplification was performed in forty-eight carcinomas and in normal breast tissues using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Primers for target genes (TGFB1 and TGFBR2) and reference gene (GAPDH) were designed by the Primers Express software (Applied Biosystems). The PCR primer sequences were: TGFB1 forward primer 5'-ggctaccatgccaacttct-3' and reverse primer 5'-tgtacaaccagcataacccgg-3' (101 pb amplicon); TGFBR2 forward primer tgggaaatgacatctcgctg-3' and reverse primer 5'-ccctgtgtcgaaagcatgaa-3' (102 pb amplicon); and GAPDH forward primer 5'-ggcctccaaggagtaagacc-3' and reverse primer 5'-aggggtctacatggcaactg-3' (147 pb amplicon).
The PCR were carried out in a total volume of 25µl according the manufacturer’s
instructions. Quantitative data was analyzed using the Sequence Detection System software (v1.0; Applied Biosystems). Standard curves for all primers were constructed using a series of 5-fold dilutions (pure, 1:5, 1:25, 1:125, 1:625) of cDNA pool (cDNA from normal and tumor breast tissues). The PCR efficiency (E) was calculated according to the equation: E=10[-1/slope] -1.The relative quantification (RQ) of the target genes in comparison to GAPDH were calculated according Pfaffl [20]. The transcripts were considered upregulated when the RQ was 2.0 and downregulated when it was 0.5.
2.4. Immunohistochemistry Staining
Formalin-fixed and paraffin-embedded tissues were freshly cut (3 µm) and the
with the primary antibody. Immunohistochemical reactions were performed using the RTU-ER-6F11 antibody (Novocastra, Newcastle, UK) (dilution 1:50), the monoclonal anti-human progesterone receptor 1A6 antibody (DAKO, Carpinteria, CA, USA) (dilution 1:50), policlonal rabbit anti-human cerbB-2 oncoprotein (DAKO, Carpinteria, CA, USA) (dilution 1:800), monoclonal mouse anti-human Ki67 antigen clone MIB-1 (DAKO, Carpinteria, CA, USA) (dilution 1:100), polyclonal rabbit antibody anti-TGF- 1 RB-10347-P (Lab Vision, Fremont, CA, USA) (dilution 1:25), polyclonal rabbit antibody anti-T RII RB-10345-P (Lab Vision, Fremont, CA, USA) (dilution 1:25). After incubation for one hour, the sections were washed in PBS, incubated for 30 min with secondary biotinylated antibody, and incubated for 30 min with streptavidin peroxidase complex (LSAB, DAKO, Carpinteria, CA, USA). The sections were developed with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. Negative and positive control slides were included with each assay. In addition, a positive control of normal placental tissue was used in each assay to quantify the TGFB1 and TGFBR2 relative expression level. Normal placental tissue stained extensively for both antibodies and the intensity was assigned as 3. It was analyzed 43 breast cancers, 31 adjacent normal breast, and two normal breast tissues. The staining intensity was scored as follow: 0 (no expression) to 3 (highest expression). Percentage of epithelial tissues expressing TGFB1 and TGFBR2 were also scored as: absent (0%), 1-24% (1), 25-49% (2), 50-74% (3), 75-100% (4). The staining intensity and percentage of positive cells product was calculated to obtain a final score. Final score was defined as follow: 0 (0 to 3); 1 (4 to 8) and 2 (9 to 12). Immunohistochemical staining scoring of for ESR1 and PGR was considered positive if the percentage of positive nuclear staining was ≥10% of neoplasic cells. In areas of
well-preserved tissue, the fraction of the infiltrating cells was scored. For HER-2, the strength of the membranous staining was recorded by a four-step scale 0, 1+ to 3+ as follows: continuous staining of membrane (3+), continuous staining present in >10% of the tumor cells (2+), focal or discontinuous staining present in >10% of tumor cells (1+), and staining in <10% of infiltrating cells (0). The Ki67 levels (percentage of stained cells) were evaluated in all cases, but eight were non-reactive. The median value of the Ki67 labeling index included in this study was used as a cutoff to distinguish tumors with low (≤25%) and high (>25%)
proliferative fractions [21]. 2.5. Statistical Analysis
presented as the median and the minimum and maximum values. Correlation analyses between immunohistochemistry (IHC) and qRT-PCR results were performed using Spearman’s rank test. Fisher’s exact test was applied to determine the strength of association between the categorical variables.
A binary logistic regression model were used to analyze the associations among TGFB1 and TGFBR2 IHC scores with the variables ESR1, PGR, and HER-2 IHC status; lymph node involvement; histological grade, and tumor size in relation to local recurrence or distant metastasis.
Disease-free survival (DFS) was calculated from the date of diagnosis to the occurrence of local recurrences or distant metastasis. Kaplan-Meier analysis was used to determine DFS according to the variables studied. The log-rank test was used to compare the differences between DFS curves.
3. Results
Most patients were over 50 years of age (65%) with mean age of 59.8 ± 15.5 (range:
30 to 94 years); 72% of the tumors were > 2 cm (pT2 and pT3), and 50% showed a low proliferation rate (Ki67 25%). Twelve cases (24%) overexpressed HER-2 protein (score 3+). During the study, two patients died, one due to unrelated cause and the other due to cancer. Two patients had BC local recurrence, six presented distant metastasis (one pleura, one bone, and four lung spread), and in three cases the follow-up was missed. A summary of the clinical and pathological data is shown in Table 1.
3.1. Transcript expression and clinical histopathological variables
By qRT-PCR, TGFB1 overexpression and downexpression were detected in a similar number of cases (14 and 11 out of 48 samples, respectively). No statistically difference was found in TGFB1 mRNA levels between tumor and normal breast tissues (P=0.90, Figure 1A). TGFBR2 mRNA expression was significantly downregulated in breast tumors compared to normal breast samples (P=0.013, Figure 1B). Low TGFBR2 transcript levels were detected in 75% of cases (RQ median value = 0.25). Only two cases showed TGFBR2 mRNA upregulated (RQ=2.59 and RQ=3.51), both with histological grade I, low S-phase fraction, negative HER-2 status and positive ESR1. It was observed a significant correlation between TGFB1 and TGFBR2 transcript levels (r=0,724, P<0.0001, data not shown).
The comparison between qRT-PCR and clinical-histopathological data are summarized in Table 2. TGFB1 transcript levels showed difference marginally significant between grade III and grade I tumors (P=0.06). In addition, TGFBR2 mRNA expression level was statistically significant in the comparison with histological groups evaluated (P=0.027). Grade III tumors showed lower expression levels than those with histological grade I (P=0.014).
Tumors in clinical staging III (TNM) showed lower expression levels of TGFB1 and TGFBR2 mRNA than stage I cases (P=0.03 and P=0.04, respectively). There were no significant associations between the expression of TGFB1 and TGFBR2 mRNAs with regional lymph nodes metastasis or local recurrences.
3.2 Protein expression and clinical-histopathological parameters
Analyses of TGFB1 and TGFBR2 protein expression and clinical-pathological parameters are shown in Table 3. The comparison between TGFB1 and TGFBR2 protein levels revealed a statistically significant correlation (r=0.51, P=0.005, data not shown). Nevertheless, none correlation was observed between TGFB1 (r=-0.11, P=0.94) and TGFBR2 (r=0.29, P=0.057) when it was compared the mRNA and protein levels. Concordance rate between qRT-PCR and IHC data was 35% (TGFB1) and 61% (TGFBR2).
TGFB1 and TGFBR2 immunostaining exhibited predominantly cytoplasmic and membranous staining patterns, respectively (Fig 2). TGFB1 and TGFBR2 proteins expression were detected in 72% (31 out of 43) and 93% (40 out of 43) of the breast tumors, respectively. Two normal breast samples presented highest level of expression for both proteins. TGFB1 protein was detected in 21 out of 31 cases (P<0.001) with lower intensity than adjacent normal samples. TGFBR2 protein expression had no significant difference between breast carcinomas and adjacent normal breast tissues (P=0.36).
TGFB1 protein expression was inversely associated with ESR1 and PGR positive tumors (P=0.021 and P=0.0076, respectively). Interestingly, the association of TGFB1 mRNA and protein levels were statistically significant in ESR1 or PGR negative cases (r=0.59, P=0.007 and r=0.63, P=0.001, respectively, data not shown).
Although not statistically significant, it was observed that patients with TGFB1 scores 0 and 1 by IHC presented recurrences (P=0.09). Interestingly, none patient with tumors expressing TGFB1 protein score 2 developed recurrence during the follow-up. A multivariate analysis was conducted to evaluate the association between TGFB1 and TGFBR2 protein expression with local recurrence or distant metastasis adjusted by presence of lymph node status; histological grade; ESR1, PGR, and HER-2 IHC status; and tumor size. TGFB1 protein level was an independent prognostic factor for distant metastasis or local recurrence (P=0.043; OR = 0.21; IC95% = 0.045 - 0.953). Further comparisons revealed no significant associations (data not shown). In addition, it was not observed association between TGFB1 and TGFBR2 protein levels with age, proliferative index, TNM stage, tumor size (pT) and HER-2 status.
3.3 Survival analysis
4. Discussion
In this study we evaluated the TGFB1 and TGFBR2 expression levels in breast tumors compared to normal breast tissues by quantitative real-time PCR and immunohistochemistry. The results showed no correlations between mRNA and protein expression levels of TGFB1 and TGFBR2 in breast samples. Concordance rate between these results was 35% for TGFB1 and 61% for TGFBR2. The comparison between IHC and mRNA expression analysis showed discrepancies already described by other studies (22-25). Similarly to our results, Soufla et al. reported a negative correlation between TGFB1 mRNA and protein expression levels [26]. Hypotheses to explain RT-PCR false negative results could be an insufficient amount of cDNA for reliable amplification, the effect of normal or inflammatory cells, or the RNA total extraction strategy that may have limited the amplification of interest transcripts. The discrepancies in the remaining cases, IHC false negative results, could reflect intratumoral heterogeneity or variation in tumor content between the tissues either for later mRNA extraction or when fixed and processed for histological examination [27]. Another possible cause for discrepancies may be due to varying translation efficiency, post-transcriptional mechanisms involved in gene regulation, or the speed of degradation of the mRNA and its protein in cancer because the protein expression is not directly proportional to its mRNA expression [28,29].
We found that TGFBR2 mRNA expression was downregulated in breast tumors in comparison to normal breast tissues. In addition, the IHC data revealed higher expression level in normal breast samples for both, TGFB1 and TGFBR2. Similarly, Gobbi et al. showed reduced TGFBR2 protein expression in breast carcinoma in comparison to normal and benign breast lesions [30]. Hinshelwood et al. evaluated the TGFBR2 mRNA expression in 18 invasive BCs, 8 BC cell lines and in 4 normal breast tissues by qRT-PCR. They found lower expression levels in all the BCs and in 88% (7 out of 8) of the BC cell lines analyzed [12].
altered by the tumoral microenvironment and, although considered as normal histopathologically, could show a different expression profile compared to breast tissues without malignancies [32].
Our findings regarding the analyses of TGFB1 and TGFBR2 levels and the clinical-hitopathological characteristics showed the lowest levels of TGFB1 and TGFBR2 transcripts in tumors with high histological grade. In breast carcinomas, it has been demonstrated a significant inverse correlation between loss of TGFBR2 protein expression with histological grade and mitotic counting [30]. However, an unexpected association was observed between high expression of TGFB1 protein and grade III tumors. A possible explanation for these findings should be a statistical bias considering that most tumors were ESR1/PGR negative with histological grade III. To evaluate this hypothesis, a multivariate analysis was performed to verify the association between histological grade and TGFB1, ESR1, PGR, and HER-2 proteins status. PGR status was the unique variable significantly associated with histological grade III compared to grade I+II breast tumors (P=0.0009, OR=0.09, CI95%=0.02-0.37, data not shown).
TGFB1 and TGFBR2 were inversely associated with proliferative index (Ki67>50%). Additionally, our results showed the lowest expression levels of TGFB1 and TGFBR2 mRNAs in stage III tumors. Primary tumors T3 (> 5 cm) presented lower TGFBR2 transcript levels than breast carcinomas T1 ( 2 cm). Previous studies showed low levels of TGFB1 mRNA [26] and TGFBR2 protein expressions [30] in stage III BCs in comparison to initial stages. In another study, the TGFB1 mRNA expression levels detected by qualitative RT-PCR were 71%, 69%, 55% e 44% in stages T1, T2, T3, and T4, respectively [33]. In overall, these data reinforce the role of TGFB1 pathway in inhibit neoplastic cells proliferation during the tumoral progression [7].
independent prognostic factor for distant metastasis or local recurrences. Conflicting results regarding the TGFB1 involvement in breast tumor progression has been published. Marrogi et al. demonstrated that TGFB1 mRNA expression was higher in patients with a favorable outcome compared to those with poor prognosis [13]. Other studies showed that high expression of TGFB1 protein was related to unfavorable prognosis [14,15]. In contrast, our findings are similar to those recently reported by Koumoundourou et al., where low expression of TGFB1 protein was related to recurrence in T1-2N0 breast tumors [16]. These data suggest that the TGFB1 could be a prognostic marker, especially in PGR negative tumors. Additional studies with higher number of patients are necessary to confirm these findings.
Concerning the relation of TGFB1 expression and ovarian hormones, our study showed a significant correlation between TGFBR2 mRNA and protein expression levels in ESR1 or PGR negative breast tumors. In addition, ESR1 or PGR positive tumors showed low TGFB1 immnostaining. Soufla et al. demonstrated a strong hormonal influence of ESR1 and PGR on TGF- mRNA expression, specifically TGFB2 and TGFB3, but not in TGFB1 [26]. Down-expression of ESR1 protein and mRNA was demonstrated 6 hours after treatment of MCF-7 breast cancer lineage with exogenous TGFB1 in culture [35]. The inhibition of ESR1 with antiestrogens can induce the secretion of TGFB1 in MCF-7 cells [36]. Similarly to our results, Koumoundourou et al. found a significant inverse association between PGR and TGFB1 protein expression [16]. We can hypothesize that in hormone responsive breast carcinomas the TGFB1 down-regulation could be a mechanism developed by the cancer cells to evade the control of proliferation promoted by TGFB1.
Acknowledgements
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Table 1. Clinical-pathological characteristics of the ductal breast cancer patients.
Variables N (%)
Age (years)
Median (range) 56 (30-94)
≤50 17 35
>50 32 65
Histological grade
I 9 18
II 14 29
III 26 53
Receptor status (IHC) ESR1
negative 20 41
positive 29 59
PGR
negative 23 47
positive 26 53
HER-2
0 30 61
1+ 3 6
2+ 4 8
3+ 12 24
Proliferative index (Ki67 status)
Low (≤25%) 18 37
High (>25%) 23 47
Non-reactive 8 16
Primary tumor size
pT1 8 16
pT2 36 74
pT3 4 8
Unknown 1 2
Regional lymph node status
pN0 25 51
pN1 13 27
pN2 7 14
pN3 4 8
Adjuvant chemotherapy
No 8 16
Yes 41 84
Adjuvant radiotherapy
No 13 27
Yes 36 73
Adjuvant tamoxifen therapy
No 17 35
Yes 32 65
Table 2. TGFB1 and TGFBR2 mRNAs expression levels according to clinical-pathological features.
TGFB1 TGFBR2
Variables N
mRNA expression levels a P mRNA expression levels a P
Histological grade *
I 8 1.85 (0.79-35.92) P1/2 0.473 0.69 (0.07-3.50) P1/2 0.161
II 14 1.13 (0.28-10.77) P1/3 0.064 0.32 (0.02-1.18) P1/3 0.014
III 26 0.77 (0.05-7.78) P2/3 0.262 0.17 (0.009-1.77) P2/3 0.136
Proliferative index (Ki67 status)
<25% 16 1.36 (0.57-35.92) P1/2 0.787 0.36 (0.08-3.50) P1/2 0.10
25-50 16 1.48 (0.39-10.77) P1/3 0.015 0.26 (0.02-1.18) P1/3 0.009
>50 8 0.62 (0.05-2.92) P2/3 0.051 0.07 (0.009-1.77) P2/3 0.061
Non-reactive 8 0.86 (0.14-2.12) 0.12 (0.01-1.56)
TNM stage
I 6 0.90 (0.45-2.92) P1/2 0.424 0.39 (0.06-1.77) P1/2 0.546
II 33 1.23 (0.05-35.92) P1/3 0.223 0.26 (0.009-3.50) P1/3 0.113
III 9 0.66 (0.20-2.35) P2/3 0.031 0.14 (0.01-0.30) P2/3 0.043
Primary tumor size
pT1 8 1.01 (0.45-2.92) P1/2 0,839 0.40 (0.06-1.77) P1/2 0,254
pT2 35 1.08 (0.05-35.92) P1/3 0.367 0.21 (0.009-3.50) P1/3 0.048
pT3 4 0.58 (0.39-3.51) P2/3 0.532 0.10 (0.02-0.26) P2/3 0.132
Lymph nodes involvement
No 24 1.17 (0.14-10.77) 0.2731 0.28 (0.02-3.50) 0.572
Yes 23 0.99 (0.05-35.92) 0.19 (0.009-2.58)
Distant metastasis / local recurrence
No 40 1.02 (0.14-10.77) 0.989 0.25 (0.01-3.50) 0.922
Yes 8 1.15 (0.05-35.92) 0.22 (0.009-2.58)
Table 3. Association between tumor expression of TGFB1 and TGFBR2 proteins revealed by immunohistochemistry assay and clinical features. TGFB1 IHC score TGFBR2 IHC score N
0 1+ 2+
P* N
0 1+ 2+
P*
Estrogen receptor 0,021 0,909
Negative 19 3 8 8 19 2 9 8
Positive 24 13 8 3 23 3 9 11
Progesterone receptor 0,007 0,912
Negative 22 5 7 10 22 2 10 10
Positive 21 11 9 1 20 3 8 9
Histological grade 0,013 0,795
I 8 5 2 1 8 2 3 3
II 13 5 8 0 12 1 6 5
III 22 6 6 10 22 2 9 11
Lymph nodes involvement 0,034 0,427
No 20 6 5 9 20 3 6 11
Yes 22 10 10 2 21 2 11 8
Regional lymph node status 0,486 0,005
pN1 12 5 6 1 11 0 4 7
pN2 7 2 4 1 7 0 6 1
pN3 3 3 0 0 3 2 1 0
Distant metastasis / local recurrence 0,099 0,298
No 36 11 14 11 35 5 13 17
Yes 7 5 2 0 7 0 5 2
