Clin Transl Oncol DOI 10.1007/s12094-014-1180-z
RESEARCH ARTICLE
Stromal expression of carbonic anhydrase IX in esophageal cancer G. Jomrich • B. Jesch • P. Birner • K. Schwameis M. Paireder • R. Asari • S. F. Schoppmann
•
Received: 9 December 2013 / Accepted: 24 March 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Purpose Carbonic anhydrase IX (CA IX), a transmembrane glycoprotein, is known as an endogenous marker for hypoxia. Overexpressed in cancer-associated fibroblasts, CA IX has been reported to be associated with a poor outcome for a number of malignant tumors. Aim of this study was to investigate the role of CA IX in the tumor surrounding stroma of esophageal cancer. Methods/patients Stromal expression of CA IX in 361 formalin-fixed, paraffin-embedded specimens of invasive esophageal cancers, 206 adenocarcinoma (AC) and 155 squamous cell carcinoma (SCC), was investigated. Results In 42 cases (11.6 %), CA IX expression in the tumor surrounding stroma (AC 23 and SCC 19) was observed. Expression of CA IX correlated with the factors tumor stage (p \ 0.001) and lymph node status (p = 0.008). Patients with CA IX expressed in the tumor surrounding stroma had a significant shorter disease-free survival (p = 0.007) and overall survival (p = 0.013). Conclusion In esophageal cancer, CA IX-expressing tumor stroma is associated with shorter survival. Inhibition of the tyrosine kinase CA IX might represent a new onset for therapies against esophageal cancer.
G. Jomrich B. Jesch K. Schwameis M. Paireder R. Asari S. F. Schoppmann (&) Gastroesophageal Cancer Unit, Department of Surgery, Upper-GI-Service, Comprehensive Cancer Center Vienna, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria e-mail: [email protected] P. Birner Clinical Institute of Pathology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria
Keywords Esophageal cancer Carbonic anhydrase IX Cancer-associated stroma Tumor micro environment (TME) Carcinoma-associated fibroblasts (CAFs)
Introduction Over decades, cancer research focused on pathobiological mechanisms in tumor cells only. Meanwhile, the tumor microenvironment came into focus of scientific interest and seems to play a major role in the progression, growth and spread of cancer [1]. Normal stroma is built by fibroblasts, immune and inflammatory cells, as well as fat cells and blood vessels. Activated by inflammation and fibrosis, fibroblasts turn into myofibroblasts, producing relevant signal mediators, such as growth factors, cytokines, chemokines and other immune modulators. After their activation and transdifferentiation, in tumors, fibroblasts are termed carcinoma-associated fibroblasts (CAFs). By expressing a variety of enzymes, CAFs promote tumor progression [2, 3]. As one of those tumorigenic enzymes, carbonic anhydrase IX (CA IX) was identified [4]. CA IX belongs to the family of zinc metalloenzymes, catalyzing the reversible hydration of carbon dioxide [5]. In normal human or other mammalian tissue, CA IX is present in the stomach, intestinal and gall bladder epithelia. In a number of different malignancies, hypoxia-related overexpression of CA IX in the tumor is associated with poor survival rates [6– 11]. Recently, CA IX expression was not only described in cancer cells but also in tumor-related stroma [12]. The prognostic significance of CA IX expression by CAFs could be shown in lung [13], colorectal [14] and breast cancer [15].
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Esophageal cancer is the ninth most frequent cancer worldwide. While the number of esophageal adenocarcinoma (AC) is increasing dramatically, the number of esophageal squamous cell carcinoma (SCC) is decreasing [16, 17]. Though improvements in therapy of esophageal cancer can be noticed, prognosis for patients is still poor [18], and targets for novel therapies are urgently needed. Recently we could show that CA IX overexpression in esophageal cancer is associated with diminished patients’ prognosis and correlates with expression of HER-2, suggesting its usability as therapeutic target [19]. Until now, no data on CA IX expression in CAF of esophageal cancer do exist. Intent of this study was to investigate the impact of CA IX expression in the surrounding tumor stroma in a large cohort of esophageal adeno and squamous cell carcinomas. The association of lymphovascular invasion (LVI), podoplanin and the signal transducer and activator of transcription 3 (pSTAT3) in esophageal cancer was recently demonstrated by our group [20–22]. Therefore, the possible interactions of CA IX with these factors and its clinical impact were investigated in this study.
Patients and methods Included in this study are all 361 patients who underwent resection of carcinomas of the esophagus or the gastroesophageal junction during the period from 1992 to 2009 at the Department of Surgery, Medical University of Vienna. Included into this study were 361 patients, 281 men and 80 women, with invasive esophageal cancer. From 206 cases of adenocarcinoma (AC) and 155 cases of squamous cell carcinoma (SCC), sufficient tissue for analysis was available. The mean age at time of surgery was 63 ± 10 years (standard deviation). In 306 (84.8 %) cases R0 resection was possible. A number of 106 (29.4 %) patients received neoadjuvant therapy. All patients received neoadjuvant treatment according to the current institutional standards. All tumors were restaged according to the UICC 7th edition TNM classification. From the analysis excluded were all cases of gastric cancer as well as all cases of Barrett’s mucosa (with or without dysplasia). Samples of either lowgrade or high-grade esophageal squamous cell dysplasia were excluded from the study as well. Immunohistochemistry (IHC) was performed on paraffin-embedded specimens fixed in 4 % buffered formalin, using 3-lm-thick histological sections. CA IX expression was detected by using a polyclonal rabbit anti-CA IX antibody (Abcam, Cambridge, UK) in a dilution of 1:1,000. Two observers (B.P. and J.G.) independently reviewed all slides. A specimen was considered as positive for expression when in C10 % of CA IX in the tumor surrounding
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stroma staining was seen. Detection of podoplanin was performed using an affinity-purified polyclonal rabbit antibody as outlined by Breiteneder-Geleff et al. [23]. The lymphovascular invasion (LVI) was estimated in all podoplanin-immunostained slides as previously described by Schoppmann et al. [24]. Nuclear expression of tyrosine-705 phosphorylated STAT3 (pY705-STAT3) was determined with a rabbit monoclonal antibody (clone D3A7, Cell Signaling Technology, Inc., Danvers, MA) in a dilution of 1:100. Immunostaining scores of pSTAT3 (IHC score: 0–300) were calculated as the products of the staining intensity (1 = weak, 2 = moderate or 3 = strong expression of pSTAT3) and the rates of staining were given in percentages of tumor cells showing positive staining (0–100 %). Tumors were considered to have high pSTAT3 expression with final scores exceeding the median score. Tumors showing expression equal or below the median were considered as low or absent as described previously [25]. Overall survival (OS) was defined as the time between primary surgery and the patient’s death. Deaths from a cause other than cancer or survival until the end of the observation period were considered as censored observations. Disease-free survival (DFS) was defined from the day of surgery until the first evidence of disease progression. Survival times were determined with Kaplan and Meier method. Significance of the differences was determined with a log-rank test. Multivariate analysis was performed with Cox proportional hazards model. Correlations between clinicopathological parameters and expression of the studied markers were analyzed with the v2 test or Fisher’s exact test. Mann–Whitney U test was used as appropriate. Statistical analysis was performed with SPSS 20.0 (IBM Corp Ó, SPSS Ó). A two-tailed p value B0.05 was considered as significant.
Results From 361 cases, 42 (11.6 %) showed C10 % CA IX expression in the tumor surrounding stroma [AC 23 (11.2 %) and SCC 19 (12.3 %)] (Fig. 1a–d). 319 (88.4 %) specimens were scored as negative or only weak positive [AC 183 (88.8 %) and SCC 136 (87.7 %)]. In two cases of complete pathologic response after preoperative chemotherapy, pre-therapeutic biopsies were applied for CA IX analysis (see Table 1). Both cases (AC) showed no CA IX expression. Pathohistological data and CA IX expression status are shown in Table 1. The correlations between the presence of CA IX in the tumor surrounding stroma and the clinicopathological parameters are compiled in Table 2. Statistical significance (p = 0.014, Chi-square test) could be shown for lymph vessel invasion (LVI) in CA IX positive adenocarcinomas. Investigating all invasive
Clin Transl Oncol
Fig. 1 a, b Specimen of esophageal adenocarcinomas. No expression of CA IX in the tumor surrounding stroma (original magnification 9100 both). c Strong stromal expression of CA IX on a specimen of an esophageal squamous cell carcinoma (original magnification
9200). d Specimen of an esophageal adenocarcinoma showing strong expression of CA IX in fibroblasts only (original magnification 9100)
cancers, no significance for LVI was found (p = 0.066, Chi-square test), as well as for squamous cell carcinomas. In all cancers a statistical significance (p \ 0.001, Chisquare test) for the correlation between the expression of CA IX and podoplanin could be found. This correlation is evident too when investigating AC (p = 0.015) and SCC (p = 0.011) separately. Significance between expression of CA IX and pSTAT3 could be shown in SCC only (p = 0.026). Correlation in the collective of all cancers and in AC missed significance with p = 0.092 and p = 1.000. In the collective of all tumors, significance for CA IX expression and the factors tumor stage (p \ 0.001, Mann– Whitney test) and lymph node status (p = 0.008, Mann– Whitney test) was found. When investigating AC and SCC separately, significance with the expression of CA IX could be shown for the factors histological tumor grading (p = 0.046), tumor stage (p = 0.006) and lymph node status (p = 0.024) in AC (all Mann–Whitney test). For SCC significance was found for the factor tumor stage only (p = 0.001, Mann–Whitney test). No correlation between
preoperative treatment and CA IX expression was found in uni- or multivariate analysis. Mean survival time was 35 months (48 month in AC and 28 in SCC). Disease recurrence occurred in 96 patients with AC and 74 patients with SCC. 82 patients with AC and 64 patients with SCC died from their cancer during this period. High CA IX expression was associated with shorter DFS (p = 0.007, log-rank test) and OS (p = 0.013, logrank test) in all tumors. In the subgroups of AC and SCC, statistically significant shorter DFS (p = 0.029, log-rank test) and OS (p = 0.045, log-rank test) could be shown for SCC only. No statistically significance for OS and DFS was found in the subgroup of AC (Fig. 2a–f). Five-year OS rates for patients with low/absent CA IX expression were 44 % (AC 47 %; SCC 39 %), and 28 % in cases with high CA IX expression (AC 42 %; SCC 10 %). Five-year DFS rates were 37 % for cases with low/absent CA IX expression (AC 41 %; SCC 33 %), and 25 % (AC 40 %; SCC 8 %) in cases with high CA IX expression. In multivariate analysis of all cancer cases including tumor stage, lymph node staging, histological grading, age,
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Clin Transl Oncol Table 1 Clinical data of cancer patients and CA IX expression Factor
All (n = 361) (%)
Adeno carcinomas (n = 206) (%)
Squamous cell carcinomas (n = 155) (%)
281 (77.80)
171 (83)
110 (71)
80 (22.20)
35 (17)
45 (29)
2 (1)
0 (0)
Sex Male Female Tumor stage ypT0a
2 (0.60)
pT1a
21 (5.80)
14 (6.80)
7 (4.50)
pT1b
41 (11.40)
21 (10.20)
20 (12.90)
pT2
95 (26.30)
58 (28.20)
37 (23.90)
pT3
185 (51.20)
103 (50.00)
82 (52.90)
pT4
17 (4.70)
8 (3.90)
9 (5.80)
Lymph node status pN0
126 (37.00)
68 (35.20)
58 (39.20)
pN1
92 (27.00)
38 (19.70)
54 (36.50)
pN2
62 (18.20)
39 (20.20)
23 (15.50)
pN3
61 (17.90)
48 (24.90)
13 (8.80)
Histological grading G1
18 (5.00)
6 (2.90)
12 (7.70)
G2 G3
190 (52.60) 153 (42.40)
87 (42.20) 113 (54.90)
103 (66.50) 40 (25.80)
CA IX expression Negative
319 (88.40)
183 (88.80)
136 (87.70)
Positive
42 (11.60)
23 (11.20)
19 (12.30)
R0 resection Yes
306 (84.80)
173 (84.00)
133 (85.80
No
55 (15.20)
33 (16.00)
22 (14.20
a
In these two cases pre-therapeutic biopsies with enough peritumoral tissue were applied for analysis (both cases CA IX negative)
R0 resection and CA IX expression, CA IX expression was not an independent prognostic factor for OS and DFS. Analyzing AC and SCC separately, CA IX expression could not be approved as an independent prognostic factor for OS and DFS as well. Statistical significance could be shown for the factors tumor stage, lymph node staging and age only (Tables 3, 4, 5).
Table 2 Correlation between CA IX in the tumor surrounding stroma and clinicopathological characteristics Factor
CA IX expression Negative (%)
p value* Positive (%)
All cancers (n = 353) LVI Negative Positive
0.066 189 (90.9) 122 (84.1)
19 (9.1) 23 (15.9)
Adenocarcinoma (n = 202) LVI
0.014
Negative
105 (93.8)
7 (6.3)
Positive
74 (82.2)
16 (17.8)
Squamous cell carcinoma (n = 151) LVI
1.000
Negative
84 (87.5)
12 (12.5)
Positive
48 (87.3)
7 (12.7)
All cancers (n = 353) \0.001
Podoplanin 0/? ??/???
245 (91.8)
22 (8.2)
66 (76.7)
20 (23.3)
Adenocarcinoma (n = 202) Podoplanin 0/?
144 (91.7)
13 (8.3)
0.015
??/???
35 (77.8)
10 (22.2)
Squamous cell carcinoma (n = 151) Podoplanin 0/? ??/???
0.011 101 (91.8)
9 (8.2)
31 (75.6)
10 (24.4)
All cancers (n = 319) pSTAT3
0.092
B10
164 (92.7)
123 (86.6)
[10
13 (7.3)
19 (13.4)
Adenocarcinoma (n = 181) pSTAT3
1.000
B10
99 (90.8)
65 (90.3)
[10
10 (9.2)
7 (9.7)
Squamous cell carcinoma (n = 138) pSTAT3 B10 [10
0.026 65 (95.6)
58 (82.9)
3 (4.4)
12 (17.1)
Discussion Despite significant improvements in the therapy of esophageal cancer over the last few years, survival rates are still unsatisfactory and new therapeutically onsets are needed urgently. To provide better therapy, it is essential to understand the pathogenetic mechanisms of esophageal cancer. Correlation between CA IX expression in cancer cells, hypoxia and survival was found in various cancers types [6–9]. Recently we could show that high CA IX
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expression in esophageal cancer is associated with shorter survival [19]. As Brockton et al. [26] for oral cavity squamous cell carcinoma and Nakao et al. [27] for lung adenocarcinoma could show, elevated stromal CA IX expression is associated with poor patients survival as well. In this study we investigated CA IX expression in the tumor surrounding stroma of 361 cases of esophageal cancer. We found that in 42 (11.6 %) of 361 cases CA IX was expressed in the tumor surrounding stroma (AC 23,
Clin Transl Oncol
Fig. 2 a Cumulative disease-free survival in all 361 patients with esophageal cancer. b Cumulative disease-free survival in 206 patients with invasive adenocarcinoma of the esophagus (AC). c Cumulative disease-free survival in 155 patients with squamous cell carcinoma of the
esophagus (SCC). d Cumulative overall survival in all 361 patients with esophageal cancer. e Cumulative overall survival in 206 patients with invasive adenocarcinoma of the esophagus (AC). f Cumulative overall survival in 155 patients with squamous cell carcinoma of the esophagus
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Clin Transl Oncol Table 3 DFS and OS with regard to clinicopathological parameters and the presence of stromal CA IX expression in all cancers
Table 4 DFS and OS with regard to clinicopathological parameters and the presence of stromal CA IX expression in adenocarcinoma
Factor
Factor
p value multivariate
Relative risk (95 % confidence interval)
All cancers
Relative risk (95 % confidence interval)
0.053 \0.001
– 1.874 (1.475–2.379)
Adenocarcinoma
DFS Staging T Staging N
p value multivariate
DFS \0.001 \0.001
1.692 (1.353–2.114) 1.586 (1.344–1.871)
Staging T Staging N
Grading
0.769
–
Grading
0.659
–
Patients age
0.036
0.984 (0.969–0.999)
Patients age
0.044
0.980 (0.960–0.999)
R0 resection
0.775
–
R0 resection
0.912
–
CA IX expression
0.081
–
CA IX expression
0.055
–
OS
OS
Staging T
\0.001
1.759 (1.379–2.244)
Staging T
0.083
Staging N
\0.001
1.543 (1.296–1.836)
Staging N
\0.001
– 1.899 (1.463–2.465)
Grading
0.0624
–
Grading
0.821
–
Patients age
0.189
–
Patients age
0.118
–
R0 resection
0.545
–
R0 resection
0.980
–
CA IX expression
0.196
–
CA IX expression
0.256
–
SCC 19). When analyzing all tumors, we found that a high expression of CA IX is associated with a significant shorter DFS and OS. These results are in good correlations with our findings we made in esophageal cancer cells previously [19]. Separating the collective significance for DFS and OS, in this study, could be found for SCC only. A statistically non-significant trend towards an increased survival for patients with CA IX negative tumors was calculated. Interestingly, this survival benefit in patients with CA IX negative tumors turned statistically significant when analyzing only patients with squamous cell carcinomas (p = 0.045). Considering these findings, one could hypothesize that the stromal expression of CA IX on OS plays a larger more important role in squamous cell carcinomas. These findings go in good accordance with previously published data by our group [19]. While tumor progression, both, cancer cells and tumor stroma are exposed to hypoxia, accordingly, we found strong correlation between CA IX and other factors, known as markers for hypoxia. The strongest relation was found between CA IX and the sialoglycoprotein podoplanin. As we could show recently, podoplanin is overexpressed in CAFs in invasive esophageal adenocarcinoma [28]. According to that previous study, we found correlations in AC between stromal CA IX expression and tumor stage (p = 0.006, Mann–Whitney test) and lymph node staging (p = 0.024, Mann–Whitney test) as well. Although we missed significance in multivariate analysis of stromal CA IX expression, the significant results for the factors tumor stage and lymph node staging underline that CA IX might influence tumor cell migration. As described in Gieling and
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Table 5 DFS and OS with regard to clinicopathological parameters and the presence of stromal CA IX expression in squamous cell carcinoma Factor
p value multivariate
Relative risk (95 % confidence interval)
Staging T
\0.001
1.994 (1.419–2.803)
Staging N
\0.001
1.624 (1.241–2.125)
Squamous cell carcinoma DFS
Grading
0.073
–
Patients age
0.425
–
R0 resection
0.428
–
CA IX expression
0.807
–
OS Staging T
\0.001
2.089 (1.440–3.055)
Staging N
0.001
1.555 (1.185–2.041)
Grading
0.293
–
Patients age
0.876
–
R0 resection CA IX expression
0.209 0.933
– –
Williams [29], there is strong evidence that acidosis promotes tumor progression. CA IX destabilizes adherent junctions between cells via modulating E-cadherin directly, after hypoxia-induced acidosis leads to increased motility, migration and neo-vascularization. The presented correlation, between CA IX and podoplanin, supports our recently discussed new field of interest, where the expression of podoplanin might support endothelial-mesenchymal transition (EndMT) by lymphatic endothelial cells [28].
Clin Transl Oncol
For cervical cancer cells, there is a CA IX-induced interface with the Rho/ROCK (Rho-associated kinase)signaling pathway [30]. As Ito et al. [31] could show the tumor promoting effect of podoplanin positive fibroblasts goes along with RhoA activity. Those two studies indicate that CA IX and podoplanin are in near relationship in promoting esophageal cancer. To our knowledge, for esophageal cancer, it is not investigated yet but might be a promising onset for new therapies. Until now, only few data exist on the stromal expression of CA IX and other hypoxia-induced factors in esophageal cancer. The observations of this underlying study underline the importance of further investigations in the field of tumor microenvironment as a potential therapeutic onset. Conflict of interest S.F. Schoppmann received an unrestricted research grant by Novartis.
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RESEARCH ARTICLE
Stromal expression of carbonic anhydrase IX in esophageal cancer G. Jomrich • B. Jesch • P. Birner • K. Schwameis M. Paireder • R. Asari • S. F. Schoppmann
•
Received: 9 December 2013 / Accepted: 24 March 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Purpose Carbonic anhydrase IX (CA IX), a transmembrane glycoprotein, is known as an endogenous marker for hypoxia. Overexpressed in cancer-associated fibroblasts, CA IX has been reported to be associated with a poor outcome for a number of malignant tumors. Aim of this study was to investigate the role of CA IX in the tumor surrounding stroma of esophageal cancer. Methods/patients Stromal expression of CA IX in 361 formalin-fixed, paraffin-embedded specimens of invasive esophageal cancers, 206 adenocarcinoma (AC) and 155 squamous cell carcinoma (SCC), was investigated. Results In 42 cases (11.6 %), CA IX expression in the tumor surrounding stroma (AC 23 and SCC 19) was observed. Expression of CA IX correlated with the factors tumor stage (p \ 0.001) and lymph node status (p = 0.008). Patients with CA IX expressed in the tumor surrounding stroma had a significant shorter disease-free survival (p = 0.007) and overall survival (p = 0.013). Conclusion In esophageal cancer, CA IX-expressing tumor stroma is associated with shorter survival. Inhibition of the tyrosine kinase CA IX might represent a new onset for therapies against esophageal cancer.
G. Jomrich B. Jesch K. Schwameis M. Paireder R. Asari S. F. Schoppmann (&) Gastroesophageal Cancer Unit, Department of Surgery, Upper-GI-Service, Comprehensive Cancer Center Vienna, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria e-mail: [email protected] P. Birner Clinical Institute of Pathology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria
Keywords Esophageal cancer Carbonic anhydrase IX Cancer-associated stroma Tumor micro environment (TME) Carcinoma-associated fibroblasts (CAFs)
Introduction Over decades, cancer research focused on pathobiological mechanisms in tumor cells only. Meanwhile, the tumor microenvironment came into focus of scientific interest and seems to play a major role in the progression, growth and spread of cancer [1]. Normal stroma is built by fibroblasts, immune and inflammatory cells, as well as fat cells and blood vessels. Activated by inflammation and fibrosis, fibroblasts turn into myofibroblasts, producing relevant signal mediators, such as growth factors, cytokines, chemokines and other immune modulators. After their activation and transdifferentiation, in tumors, fibroblasts are termed carcinoma-associated fibroblasts (CAFs). By expressing a variety of enzymes, CAFs promote tumor progression [2, 3]. As one of those tumorigenic enzymes, carbonic anhydrase IX (CA IX) was identified [4]. CA IX belongs to the family of zinc metalloenzymes, catalyzing the reversible hydration of carbon dioxide [5]. In normal human or other mammalian tissue, CA IX is present in the stomach, intestinal and gall bladder epithelia. In a number of different malignancies, hypoxia-related overexpression of CA IX in the tumor is associated with poor survival rates [6– 11]. Recently, CA IX expression was not only described in cancer cells but also in tumor-related stroma [12]. The prognostic significance of CA IX expression by CAFs could be shown in lung [13], colorectal [14] and breast cancer [15].
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Esophageal cancer is the ninth most frequent cancer worldwide. While the number of esophageal adenocarcinoma (AC) is increasing dramatically, the number of esophageal squamous cell carcinoma (SCC) is decreasing [16, 17]. Though improvements in therapy of esophageal cancer can be noticed, prognosis for patients is still poor [18], and targets for novel therapies are urgently needed. Recently we could show that CA IX overexpression in esophageal cancer is associated with diminished patients’ prognosis and correlates with expression of HER-2, suggesting its usability as therapeutic target [19]. Until now, no data on CA IX expression in CAF of esophageal cancer do exist. Intent of this study was to investigate the impact of CA IX expression in the surrounding tumor stroma in a large cohort of esophageal adeno and squamous cell carcinomas. The association of lymphovascular invasion (LVI), podoplanin and the signal transducer and activator of transcription 3 (pSTAT3) in esophageal cancer was recently demonstrated by our group [20–22]. Therefore, the possible interactions of CA IX with these factors and its clinical impact were investigated in this study.
Patients and methods Included in this study are all 361 patients who underwent resection of carcinomas of the esophagus or the gastroesophageal junction during the period from 1992 to 2009 at the Department of Surgery, Medical University of Vienna. Included into this study were 361 patients, 281 men and 80 women, with invasive esophageal cancer. From 206 cases of adenocarcinoma (AC) and 155 cases of squamous cell carcinoma (SCC), sufficient tissue for analysis was available. The mean age at time of surgery was 63 ± 10 years (standard deviation). In 306 (84.8 %) cases R0 resection was possible. A number of 106 (29.4 %) patients received neoadjuvant therapy. All patients received neoadjuvant treatment according to the current institutional standards. All tumors were restaged according to the UICC 7th edition TNM classification. From the analysis excluded were all cases of gastric cancer as well as all cases of Barrett’s mucosa (with or without dysplasia). Samples of either lowgrade or high-grade esophageal squamous cell dysplasia were excluded from the study as well. Immunohistochemistry (IHC) was performed on paraffin-embedded specimens fixed in 4 % buffered formalin, using 3-lm-thick histological sections. CA IX expression was detected by using a polyclonal rabbit anti-CA IX antibody (Abcam, Cambridge, UK) in a dilution of 1:1,000. Two observers (B.P. and J.G.) independently reviewed all slides. A specimen was considered as positive for expression when in C10 % of CA IX in the tumor surrounding
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stroma staining was seen. Detection of podoplanin was performed using an affinity-purified polyclonal rabbit antibody as outlined by Breiteneder-Geleff et al. [23]. The lymphovascular invasion (LVI) was estimated in all podoplanin-immunostained slides as previously described by Schoppmann et al. [24]. Nuclear expression of tyrosine-705 phosphorylated STAT3 (pY705-STAT3) was determined with a rabbit monoclonal antibody (clone D3A7, Cell Signaling Technology, Inc., Danvers, MA) in a dilution of 1:100. Immunostaining scores of pSTAT3 (IHC score: 0–300) were calculated as the products of the staining intensity (1 = weak, 2 = moderate or 3 = strong expression of pSTAT3) and the rates of staining were given in percentages of tumor cells showing positive staining (0–100 %). Tumors were considered to have high pSTAT3 expression with final scores exceeding the median score. Tumors showing expression equal or below the median were considered as low or absent as described previously [25]. Overall survival (OS) was defined as the time between primary surgery and the patient’s death. Deaths from a cause other than cancer or survival until the end of the observation period were considered as censored observations. Disease-free survival (DFS) was defined from the day of surgery until the first evidence of disease progression. Survival times were determined with Kaplan and Meier method. Significance of the differences was determined with a log-rank test. Multivariate analysis was performed with Cox proportional hazards model. Correlations between clinicopathological parameters and expression of the studied markers were analyzed with the v2 test or Fisher’s exact test. Mann–Whitney U test was used as appropriate. Statistical analysis was performed with SPSS 20.0 (IBM Corp Ó, SPSS Ó). A two-tailed p value B0.05 was considered as significant.
Results From 361 cases, 42 (11.6 %) showed C10 % CA IX expression in the tumor surrounding stroma [AC 23 (11.2 %) and SCC 19 (12.3 %)] (Fig. 1a–d). 319 (88.4 %) specimens were scored as negative or only weak positive [AC 183 (88.8 %) and SCC 136 (87.7 %)]. In two cases of complete pathologic response after preoperative chemotherapy, pre-therapeutic biopsies were applied for CA IX analysis (see Table 1). Both cases (AC) showed no CA IX expression. Pathohistological data and CA IX expression status are shown in Table 1. The correlations between the presence of CA IX in the tumor surrounding stroma and the clinicopathological parameters are compiled in Table 2. Statistical significance (p = 0.014, Chi-square test) could be shown for lymph vessel invasion (LVI) in CA IX positive adenocarcinomas. Investigating all invasive
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Fig. 1 a, b Specimen of esophageal adenocarcinomas. No expression of CA IX in the tumor surrounding stroma (original magnification 9100 both). c Strong stromal expression of CA IX on a specimen of an esophageal squamous cell carcinoma (original magnification
9200). d Specimen of an esophageal adenocarcinoma showing strong expression of CA IX in fibroblasts only (original magnification 9100)
cancers, no significance for LVI was found (p = 0.066, Chi-square test), as well as for squamous cell carcinomas. In all cancers a statistical significance (p \ 0.001, Chisquare test) for the correlation between the expression of CA IX and podoplanin could be found. This correlation is evident too when investigating AC (p = 0.015) and SCC (p = 0.011) separately. Significance between expression of CA IX and pSTAT3 could be shown in SCC only (p = 0.026). Correlation in the collective of all cancers and in AC missed significance with p = 0.092 and p = 1.000. In the collective of all tumors, significance for CA IX expression and the factors tumor stage (p \ 0.001, Mann– Whitney test) and lymph node status (p = 0.008, Mann– Whitney test) was found. When investigating AC and SCC separately, significance with the expression of CA IX could be shown for the factors histological tumor grading (p = 0.046), tumor stage (p = 0.006) and lymph node status (p = 0.024) in AC (all Mann–Whitney test). For SCC significance was found for the factor tumor stage only (p = 0.001, Mann–Whitney test). No correlation between
preoperative treatment and CA IX expression was found in uni- or multivariate analysis. Mean survival time was 35 months (48 month in AC and 28 in SCC). Disease recurrence occurred in 96 patients with AC and 74 patients with SCC. 82 patients with AC and 64 patients with SCC died from their cancer during this period. High CA IX expression was associated with shorter DFS (p = 0.007, log-rank test) and OS (p = 0.013, logrank test) in all tumors. In the subgroups of AC and SCC, statistically significant shorter DFS (p = 0.029, log-rank test) and OS (p = 0.045, log-rank test) could be shown for SCC only. No statistically significance for OS and DFS was found in the subgroup of AC (Fig. 2a–f). Five-year OS rates for patients with low/absent CA IX expression were 44 % (AC 47 %; SCC 39 %), and 28 % in cases with high CA IX expression (AC 42 %; SCC 10 %). Five-year DFS rates were 37 % for cases with low/absent CA IX expression (AC 41 %; SCC 33 %), and 25 % (AC 40 %; SCC 8 %) in cases with high CA IX expression. In multivariate analysis of all cancer cases including tumor stage, lymph node staging, histological grading, age,
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Clin Transl Oncol Table 1 Clinical data of cancer patients and CA IX expression Factor
All (n = 361) (%)
Adeno carcinomas (n = 206) (%)
Squamous cell carcinomas (n = 155) (%)
281 (77.80)
171 (83)
110 (71)
80 (22.20)
35 (17)
45 (29)
2 (1)
0 (0)
Sex Male Female Tumor stage ypT0a
2 (0.60)
pT1a
21 (5.80)
14 (6.80)
7 (4.50)
pT1b
41 (11.40)
21 (10.20)
20 (12.90)
pT2
95 (26.30)
58 (28.20)
37 (23.90)
pT3
185 (51.20)
103 (50.00)
82 (52.90)
pT4
17 (4.70)
8 (3.90)
9 (5.80)
Lymph node status pN0
126 (37.00)
68 (35.20)
58 (39.20)
pN1
92 (27.00)
38 (19.70)
54 (36.50)
pN2
62 (18.20)
39 (20.20)
23 (15.50)
pN3
61 (17.90)
48 (24.90)
13 (8.80)
Histological grading G1
18 (5.00)
6 (2.90)
12 (7.70)
G2 G3
190 (52.60) 153 (42.40)
87 (42.20) 113 (54.90)
103 (66.50) 40 (25.80)
CA IX expression Negative
319 (88.40)
183 (88.80)
136 (87.70)
Positive
42 (11.60)
23 (11.20)
19 (12.30)
R0 resection Yes
306 (84.80)
173 (84.00)
133 (85.80
No
55 (15.20)
33 (16.00)
22 (14.20
a
In these two cases pre-therapeutic biopsies with enough peritumoral tissue were applied for analysis (both cases CA IX negative)
R0 resection and CA IX expression, CA IX expression was not an independent prognostic factor for OS and DFS. Analyzing AC and SCC separately, CA IX expression could not be approved as an independent prognostic factor for OS and DFS as well. Statistical significance could be shown for the factors tumor stage, lymph node staging and age only (Tables 3, 4, 5).
Table 2 Correlation between CA IX in the tumor surrounding stroma and clinicopathological characteristics Factor
CA IX expression Negative (%)
p value* Positive (%)
All cancers (n = 353) LVI Negative Positive
0.066 189 (90.9) 122 (84.1)
19 (9.1) 23 (15.9)
Adenocarcinoma (n = 202) LVI
0.014
Negative
105 (93.8)
7 (6.3)
Positive
74 (82.2)
16 (17.8)
Squamous cell carcinoma (n = 151) LVI
1.000
Negative
84 (87.5)
12 (12.5)
Positive
48 (87.3)
7 (12.7)
All cancers (n = 353) \0.001
Podoplanin 0/? ??/???
245 (91.8)
22 (8.2)
66 (76.7)
20 (23.3)
Adenocarcinoma (n = 202) Podoplanin 0/?
144 (91.7)
13 (8.3)
0.015
??/???
35 (77.8)
10 (22.2)
Squamous cell carcinoma (n = 151) Podoplanin 0/? ??/???
0.011 101 (91.8)
9 (8.2)
31 (75.6)
10 (24.4)
All cancers (n = 319) pSTAT3
0.092
B10
164 (92.7)
123 (86.6)
[10
13 (7.3)
19 (13.4)
Adenocarcinoma (n = 181) pSTAT3
1.000
B10
99 (90.8)
65 (90.3)
[10
10 (9.2)
7 (9.7)
Squamous cell carcinoma (n = 138) pSTAT3 B10 [10
0.026 65 (95.6)
58 (82.9)
3 (4.4)
12 (17.1)
Discussion Despite significant improvements in the therapy of esophageal cancer over the last few years, survival rates are still unsatisfactory and new therapeutically onsets are needed urgently. To provide better therapy, it is essential to understand the pathogenetic mechanisms of esophageal cancer. Correlation between CA IX expression in cancer cells, hypoxia and survival was found in various cancers types [6–9]. Recently we could show that high CA IX
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expression in esophageal cancer is associated with shorter survival [19]. As Brockton et al. [26] for oral cavity squamous cell carcinoma and Nakao et al. [27] for lung adenocarcinoma could show, elevated stromal CA IX expression is associated with poor patients survival as well. In this study we investigated CA IX expression in the tumor surrounding stroma of 361 cases of esophageal cancer. We found that in 42 (11.6 %) of 361 cases CA IX was expressed in the tumor surrounding stroma (AC 23,
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Fig. 2 a Cumulative disease-free survival in all 361 patients with esophageal cancer. b Cumulative disease-free survival in 206 patients with invasive adenocarcinoma of the esophagus (AC). c Cumulative disease-free survival in 155 patients with squamous cell carcinoma of the
esophagus (SCC). d Cumulative overall survival in all 361 patients with esophageal cancer. e Cumulative overall survival in 206 patients with invasive adenocarcinoma of the esophagus (AC). f Cumulative overall survival in 155 patients with squamous cell carcinoma of the esophagus
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Clin Transl Oncol Table 3 DFS and OS with regard to clinicopathological parameters and the presence of stromal CA IX expression in all cancers
Table 4 DFS and OS with regard to clinicopathological parameters and the presence of stromal CA IX expression in adenocarcinoma
Factor
Factor
p value multivariate
Relative risk (95 % confidence interval)
All cancers
Relative risk (95 % confidence interval)
0.053 \0.001
– 1.874 (1.475–2.379)
Adenocarcinoma
DFS Staging T Staging N
p value multivariate
DFS \0.001 \0.001
1.692 (1.353–2.114) 1.586 (1.344–1.871)
Staging T Staging N
Grading
0.769
–
Grading
0.659
–
Patients age
0.036
0.984 (0.969–0.999)
Patients age
0.044
0.980 (0.960–0.999)
R0 resection
0.775
–
R0 resection
0.912
–
CA IX expression
0.081
–
CA IX expression
0.055
–
OS
OS
Staging T
\0.001
1.759 (1.379–2.244)
Staging T
0.083
Staging N
\0.001
1.543 (1.296–1.836)
Staging N
\0.001
– 1.899 (1.463–2.465)
Grading
0.0624
–
Grading
0.821
–
Patients age
0.189
–
Patients age
0.118
–
R0 resection
0.545
–
R0 resection
0.980
–
CA IX expression
0.196
–
CA IX expression
0.256
–
SCC 19). When analyzing all tumors, we found that a high expression of CA IX is associated with a significant shorter DFS and OS. These results are in good correlations with our findings we made in esophageal cancer cells previously [19]. Separating the collective significance for DFS and OS, in this study, could be found for SCC only. A statistically non-significant trend towards an increased survival for patients with CA IX negative tumors was calculated. Interestingly, this survival benefit in patients with CA IX negative tumors turned statistically significant when analyzing only patients with squamous cell carcinomas (p = 0.045). Considering these findings, one could hypothesize that the stromal expression of CA IX on OS plays a larger more important role in squamous cell carcinomas. These findings go in good accordance with previously published data by our group [19]. While tumor progression, both, cancer cells and tumor stroma are exposed to hypoxia, accordingly, we found strong correlation between CA IX and other factors, known as markers for hypoxia. The strongest relation was found between CA IX and the sialoglycoprotein podoplanin. As we could show recently, podoplanin is overexpressed in CAFs in invasive esophageal adenocarcinoma [28]. According to that previous study, we found correlations in AC between stromal CA IX expression and tumor stage (p = 0.006, Mann–Whitney test) and lymph node staging (p = 0.024, Mann–Whitney test) as well. Although we missed significance in multivariate analysis of stromal CA IX expression, the significant results for the factors tumor stage and lymph node staging underline that CA IX might influence tumor cell migration. As described in Gieling and
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Table 5 DFS and OS with regard to clinicopathological parameters and the presence of stromal CA IX expression in squamous cell carcinoma Factor
p value multivariate
Relative risk (95 % confidence interval)
Staging T
\0.001
1.994 (1.419–2.803)
Staging N
\0.001
1.624 (1.241–2.125)
Squamous cell carcinoma DFS
Grading
0.073
–
Patients age
0.425
–
R0 resection
0.428
–
CA IX expression
0.807
–
OS Staging T
\0.001
2.089 (1.440–3.055)
Staging N
0.001
1.555 (1.185–2.041)
Grading
0.293
–
Patients age
0.876
–
R0 resection CA IX expression
0.209 0.933
– –
Williams [29], there is strong evidence that acidosis promotes tumor progression. CA IX destabilizes adherent junctions between cells via modulating E-cadherin directly, after hypoxia-induced acidosis leads to increased motility, migration and neo-vascularization. The presented correlation, between CA IX and podoplanin, supports our recently discussed new field of interest, where the expression of podoplanin might support endothelial-mesenchymal transition (EndMT) by lymphatic endothelial cells [28].
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For cervical cancer cells, there is a CA IX-induced interface with the Rho/ROCK (Rho-associated kinase)signaling pathway [30]. As Ito et al. [31] could show the tumor promoting effect of podoplanin positive fibroblasts goes along with RhoA activity. Those two studies indicate that CA IX and podoplanin are in near relationship in promoting esophageal cancer. To our knowledge, for esophageal cancer, it is not investigated yet but might be a promising onset for new therapies. Until now, only few data exist on the stromal expression of CA IX and other hypoxia-induced factors in esophageal cancer. The observations of this underlying study underline the importance of further investigations in the field of tumor microenvironment as a potential therapeutic onset. Conflict of interest S.F. Schoppmann received an unrestricted research grant by Novartis.
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