Systematic Analysis of CA9 as a Pan-Cancer Marker for Prognosis and Immunity

Well-differentiated pancreatic neuroendocrine neoplasms/tumors (PanNETs) are rare neoplasms with diverse clinical behavior. Biomarker discovery is important for predicting clinical course and prognosis of PanNET patients. Carbonic anhydrase 9 (CA9) and vimentin are hypoxia and epithelial-mesenchymal transition-related proteins of which expression in many carcinomas has been associated with poor prognosis, but their significance in PanNET has yet to be determined. We assessed CA9 and vimentin expression in 164 PanNETs and compared this with clinicopathologic characteristics. CA9 expression was observed in normal islets, while neuroendocrine microadenomas and small (< 1cm) PanNETs showed loss of CA9 expression. CA9 and vimentin expression was observed in 38 (23%) and 36 (22%) of PanNETs, respectively. CA9 expression was associated with larger size (p = 0.001), higher grade (p < 0.001), higher pT category (p < 0.001), lymph node (p = 0.003) and distant (p = 0.047) metastases, higher AJCC stage (p < 0.001), and lymphovascular (p < 0.001) and perineural (p = 0.002) invasion. PanNET patients with CA9 expression had a shorter recurrence-free survival (5-year survival rate 47%) than those without CA9 expression (76%) by univariate (p = 0.001) but not multivariate analysis. Vimentin expression correlated with CA9 expression (p < 0.001) but not with other clinicopathologic factors. In conclusion, CA9 expression was observed in normal islets, while neuroendocrine microadenomas and small (< 1cm) PanNETs showed CA9 expression loss. CA9 expression gradually reappeared in larger PanNETs, and this was associated with clinical progression and decreased patient survival by univariate but not multivariate analysis.

BackgroundccRCC is frequently driven by VHL loss, leading to HIF-2α stabilization and upregulation of downstream targets such as carbonic anhydrase 9 (CA9). While CA9 is a well-established diagnostic marker of HIF pathway activation, its prognostic and predictive roles remain unclear. Emerging evidence suggests that high CA9 and HIF-2α expression may be associated with improved outcomes in ccRCC. With the recent approval of the HIF-2α inhibitor belzutifan, understanding whether these markers predict treatment response is critical for guiding biomarker-driven therapy.MethodsNext-generation sequencing of DNA (592-gene panel or whole exome) and RNA (whole transcriptome) were performed on ccRCC specimens through Caris Life Sciences to comprehensively characterize genomic and transcriptomic alterations. Expression levels of CA9 and HIF-2α were quantified based on RNA transcripts per million (TPM) and categorized as High or Low using the >75th vs < 25th percentile cutoff. Overall survival (OS) was defined as the time from initial diagnosis to death or last known follow-up. Time on treatment (TOT) was defined as the duration from the start of systemic therapy to treatment discontinuation.ResultsA total of 764 ccRCC specimens were analyzed. Of these, 433 (56.7%) were derived from primary kidney tumors, while the remaining samples were from metastatic sites, including lung (n = 71, 9.3%), bone (n = 70, 9.2%), endocrine (n = 43, 5.6%), liver (n = 23, 3%), lymph node (n = 23, 3%), CNS (n = 19, 2.5%), GI (n = 4, 0.5%) and other sites (n = 78, 10.2%). VHL alterations occurred in 82% (626/764) ccRCC tumors. The median age was 63. CA9 and HIF-2α expression levels were relatively higher in primary kidney tumors compared to metastatic sites. Expression levels of CA9 and HIF-2α were comparable across racial and ethnic subgroups. High CA9 expression (Q4 vs Q1) was associated with improved OS (mOS: 95 vs 42 months; HR 0.55, P = .005). Similarly, high HIF-2α expression (Q4 vs Q1) correlated with longer survival (mOS: 54.8 vs 38.3 months; HR 0.56, P < .001). Among belzutifan-treated patients (n = 80), CA9 expression was not associated with OS (mOS: NE vs 17.3, p : 0.851) or TOT of belzutifan (mTOT: 3.49 vs 2.34, p: 0.175). HIF-2α expression was not associated with OS (mOS: 16.2 vs 13.4, p : 0.89) or TOT of belzutifan among VHL mutant patients (2.96 vs 2.96; p: 0.91). There were limited VHL wild-type ccRCC patients (n = 9) treated with belzutifan in our cohort.ConclusionsThis comprehensive analysis of 764 ccRCC specimens demonstrates that VHL alterations are present in the majority of ccRCC tumors. CA9 and HIF-2α expression were consistent across racial and ethnic groups but showed higher expression in primary tumors compared to metastatic sites. High expression of both CA9 and HIF-2α were associated with significantly improved overall survival in the general ccRCC population. However, neither CA9 nor HIF-2α expression levels predicted response to belzutifan therapy. These findings enhance our understanding of HIF pathway biology in ccRCC and provide important context for the potential clinical application of CA9 PET imaging and HIF-2α inhibitor therapy in diverse patient populations.

Carbonic anhydrase 9 (CA9) is a plasma membrane-associated isoenzyme that catalyzes pH regulation under hypoxic conditions. CA9 is transcriptionally regulated by hypoxia-inducible factor 1. Recent studies reported that hypoxia also promoted the epithelial-mesenchymal transition (EMT) in various cancers. In the present study, we evaluated the relationship between CA9 expression and EMT invitro with two hepatoma cell lines. We also examined the clinical significance of CA9 expression in 117consecutive patients that underwent hepatectomies for hepatocellular carcinoma (HCC). We evaluated CA9 expression and EMT induction under hypoxia with quantitative RT-PCR, western blot analysis and immunofluorescence staining, in HuH7 and HepG2 cells. We knocked down CA9 expression with small interfering RNA to evaluate the relationship between CA9 and EMT. We found that hypoxia induced CA9 expression in HCC cells and promoted EMT, evidenced by a loss of E-cadherin and an increase in N-cadherin. Twist, a transcriptional regulator of EMT, was also upregulated with hypoxia. The CA9 deficiency attenuated hypoxia-induced changes in E-cadherin and N-cadherin. Immunohistochemical evaluations of patient samples showed that CA9 was expressed in 50.4% of patients (59/117). However, patients with and without CA9 expression were not significantly different in clinicopathological factors. Nevertheless, a multivariate analysis showed that CA9 expression was an independent factor for both recurrence and prognosis among patients that underwent curative surgery for HCC. In conclusion, this study revealed that CA9 expression was a pivotal predictive factor for poor prognosis after radical surgery for HCC. Moreover, the CA9 regulation of the expression of EMT-related molecules represented a mechanism that enhanced malignant potential.

Assessment of microvessel density and carbonic anhydrase-9 (CA-9) expression in rectal cancer

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia

Gallbladder cancer (GBC) is an aggressive tumour that is usually diagnosed at advanced stages and is characterised by a poor prognosis. Using public data of normal human tissues, we found that mRNA and protein levels of mucin 5B (MUC5B) and carbonic anhydrase 9 (CA9) were highly increased in gallbladder tissues. In addition, previous evidence has shown that claudin 18 (CLDN18) protein expression is higher in GBC. The aim of this study was to perform an analysis of these cell surface proteins during the histological progression of GBC in order to identify their theranostic potential. MUC5B expression, CA9 expression and CLDN18 expression were examined by immunohistochemistry in a series of 179 chronic cholecystitis (including 16 metaplastic tissues), 15 dysplasia and 217 GBC samples by the use of tissue microarray analysis. A composite staining score was calculated from staining intensity and percentage of positive cells. Immunohistochemical analysis showed high expression of MUC5B and CA9 among normal epithelium, metaplastic tissues, and dysplastic tissues. However, expression of both proteins was observed in roughly 50% of GBC samples. In contrast, CLDN18 was absent in normal epithelium, but its expression was higher in metaplastic cells. Among GBC cases, approximately half showed high CLDN18 expression. No associations were found between MUC5B, CA9 and CLDN18 expression and any clinicopathological features. CLDN18 is a new metaplasia marker in gallbladder tissues, and is conserved in approximately half of GBC cases. MUC5B and CA9 are highly conserved during GBC histological progression. The three markers are potential theranostic markers, in particular CA9 and CLDN18, for which there are already targeted therapies available.

Background: In HCC, hypoxia and MET can promote tumor progression and induce resistance to radiation, chemo or targeted therapies. The aim of this study was to correlate MET and hypoxia marker carbonic anhydrase 9 (CA9) expression levels by immunohistochemistry with clinicopathological characteristics and disease-free survival (DFS) in patients with HCC. Material and methods: One-hundred HCC resection specimens were evaluated by immunohistochemistry for MET (clone sp44, Ventana) and CA9 (rabbit polyclonal) expression. For automated evaluation, we elaborated our own macro using ImageJ software, and compared it with H- and MetMab validated scores. METhigh and CA9high expression were defined as moderate to strong staining. Staining results were correlated with clinicopathological characteristics. Univariate analyses were performed using Fisher's exact or chi square tests, and multivariate analyses using Cox regression model. Median DFS (mDFS) were calculated by Kaplan-Meier method. MET amplification was assessed by FISH (zytovision probe) and centromere 7 (CEN7) used as an internal control. Amplification was defined as a mean ratio of MET/CEN7&amp;gt; 4, counting 100 nuclei. Results: Tumors were BCLC A1 (92%), uninodular (53%), moderately differentiated (63%), with pathological vascular invasion (71%), and had low AFP expression (82%). METhigh expression was observed in 53% of tumors being higher in patients with viral hepatitis-associated HCC (p = 0.02) and in patients with AFP&amp;gt;400UI/L (p = 0.03). CA9high expression was observed in 41% of tumors and was correlated with viral hepatitis (p = 0.002), pathological vascular invasion (p = 0.007), and poor differentiation (p = 0.007). MET and CA9 expression levels significantly correlated with each other (p = 0.008). mDFS was shorter in METhigh (12.9 vs &amp;gt;80 months in METlow patients; p = 0.018) and CA9high (10.2 vs 34.4 months in CA9low patients; p = 0.02) populations. Combination of MET and CA9 status discriminated 3 prognostic groups. In the METhigh/CA9high group mDFS was 10.7 months, whereas mDFS was not reached in the METlow/CA9low group (p = 0.003). Using H- and MetMab scores, mDFS of the METhigh group were 12.9 months and 12.4 months respectively, and not reached in the METlow group. The METhigh/CA9low and METlow/CA9high groups were defined as intermediate risk populations (mDFS = 19 months). In multivariate analysis, tumor &amp;lt; 5cm (p = 0.025), uninodular morphology (p = 0.004), and CA9-low levels (p = 0.007) were independently associated with prolonged DFS. METhigh expression level was associated with shorter DFS when CA9 was excluded from the model (p = 0.024). No amplification was detected in METhigh HCC samples. Conclusion: MET expression is a useful prognostic marker in HCC. Patients with HCC overexpressing MET and CA9 represent a subgroup with poor prognosis that might benefit from MET inhibition therapy. Citation Format: Annemilai Tijeras-Raballand, Cindy Neuzillet, Miguel Albuquerque, Nathalie Colnot, Friedhelm Bladt, Manfred Klevesath, Christian Ihling, Hongxia Zheng, Maryse Baia, Christiane Copie-Bergman, Eric Raymond, Armand de Gramont, Valérie Paradis, Sandrine Faivre. High MET and CA9 expressions define a subgroup of hepatocellular carcinoma (HCC) patients with poor prognosis candidates for MET inhibition strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1911. doi:10.1158/1538-7445.AM2015-1911

Clinical impact of carbonic anhydrase 9 expression on neoadjuvant chemoradiotherapy in pancreatic ductal adenocarcinoma

Carbonic anhydrase 9 in clear cell renal cell carcinoma: A marker for diagnosis, prognosis and treatment

BackgroundHypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy. Therefore, identifying hypoxic environments within tumours is extremely useful for understanding cancer biology and developing novel therapeutic strategies. Several studies have suggested that carbonic anhydrase 9 (CA9) is a reliable biomarker of hypoxia and a potential therapeutic target, while pimonidazole has been identified as an exogenous hypoxia marker. However, other studies have suggested that CA9 expression is not directly induced by hypoxia and it is not expressed in all types of tumours. Thus, in this study, we focused on endoplasmic reticulum disulphide oxidase 1α (ERO1α), a protein that localises in the endoplasmic reticulum and is involved in the formation of disulphide bonds in proteins, to determine whether it could serve as a potential tumour-hypoxia biomarker.MethodsUsing quantitative real-time polymerase chain reaction, we analysed the mRNA expression of ERO1α and CA9 in different normal and cancer cell lines. We also determined the protein expression levels of ERO1α and CA9 in these cell lines by western blotting. We then investigated the hypoxia-inducible ERO1α and CA9 expression and localisation in HCT116 and HeLa cells, which express low (CA9-low) and high (CA9-high) levels of CA9, respectively. A comparative analysis was performed using pimonidazole, an exogenous hypoxic marker, as a positive control. The expression and localisation of ERO1α and CA9 in tumour spheres during hypoxia were analysed by a tumour sphere formation assay. Finally, we used a mouse model to investigate the localisation of ERO1α and CA9 in tumour xenografts using several cell lines.ResultsWe found that ERO1α expression increased under chronic hypoxia. Our results show that ERO1α was hypoxia-induced in all the tested cancer cell lines. Furthermore, in the comparative analysis using CA9 and pimonidazole, ERO1α had a similar localisation to pimonidazole in both CA9-low and CA9-high cell lines.ConclusionERO1α can serve as a novel endogenous chronic hypoxia marker that is more reliable than CA9 and can be used as a diagnostic biomarker and therapeutic target for cancer.

Carbonic anhydrase 9 (CA9), as one of the most hypoxia-responsive genes, has been associated almost exclusively with hypoxic tumors. Its principal role is in pH regulation which helps tumor cells overcome intracellular acidosis and survive extended periods of time with low oxygen. Hypoxia-inducible factor 1 (HIF-1) is the main transcriptional activator of CA9. Hepatitis B virus X protein (HBx) has been shown to increase the transcriptional activity of HIF-1. HBx is often expressed from the gene integrated in the hepatocytes infected persistently and contributes significantly to alterations in host gene expression that can lead to the development of hepatocellular carcinoma (HCC) associated with Hepatitis B virus (HBV). The aim of this study was to determine the effect of HBx on expression of CA9. Transient transfection of HBx led to an increase in the expression of CA9 as assessed by RT-PCR and Western blotting. HBx was able to increase CA9 promoter activity significantly in several cell lines. The effect was mediated via HIF-1 and a functional HRE element located -10/-3 bp upstream of the CA9 transcription initiation site. These data suggest that CA9 may be involved in the development of HCC by contributing to the survival of hepatocytes infected with HBV in liver tissue with fibrosis.

Carbonic anhydrase 9 (CA9) is a glycoprotein present on the surface of cell membranes. It is expressed in 90% of renal cancer cells, but not in normal kidney tissue. Immunotherapy targeting CA9 is underway, and our group has also conducted a clinical trial using CA9 as a cancer vaccine and confirmed the induction of cytotoxic T lymphocytes, with efficacy in some cases. Expression of CA9 antigen in oral cancer has not been reported in Japan, but our results indicate that immunotherapy targeting CA9 might be possible. We immunohistochemically observed the expression of antigens such as CA9, Ki-67, glucose transporter-1 (GLUT-1) and p53 in 107 subjects with oral squamous cell carcinoma, and examined their correlation with clinicopathological parameters. Immunostaining analysis showed expression of CA9 in 98% of oral cancer subjects, and the survival rate was significantly lower in subjects with CA9 antigen expression in 50% or more cells (P<0.05). Subjects with poorly differentiated, T4 and lymph node metastasis, or Stage IV cancer with high CA9 expression (≥50%) had a worse outcome than those with low CA9 expression. Although GLUT-1 expression was observed in 98% of subjects, similarly to CA9 expression, no significant correlation between its expression and the survival rate was seen. However, subjects with lymph node metastasis had significantly higher GLUT-1 expression, demonstrating that GLUT-1 could be an indicator of lymph node metastasis. Ki-67 was expressed in 92% of subjects, but no correlation with outcome was observed. Expression of p53 was noted in 78% of subjects, and it was found that many oral cancers have p53 genetic abnormalities, but no correlation between p53 and outcome was observed. It was confirmed that CA9 antigen is expressed in most oral cancer subjects, suggesting the possibility of immunotherapy targeting CA9 antigen in oral cancer.

Tumor cells undergo a variety of biological changes under sustained hypoxic conditions, allowing cells to survive and retain their clonogenic potential. The purpose of this study is to relate the expression of the hypoxia marker carbonic anhydrase 9 (CA9) to the uptake of iododeoxyuridine (IdUrd), a marker of proliferation, in head and neck squamous cell carcinomas. Colocalization of IdUrd and CA9 may identify an important subpopulation of tumor cells that might be responsible for repopulation and disease progression. Expression of CA9, IdUrd labeling, and colocalization between IdUrd and CA9 was examined by immunohistochemistry in biopsies of head and neck squamous cell carcinomas. Biopsies were taken from 51 patients recruited between 1998 and 2001 after administration of the proliferation marker IdUrd. A large variation was observed between the tumors in CA9 expression (range 0-39%), IdUrd labeling (range 0-81%), and colocalization between IdUrd and CA9 [FId(CA9); range 0-53%]. FId(CA9), the fraction of IdUrd-labeled cells positive for CA9, was highest at an intermediate distance from the blood vessels (100-150 microm). IdUrd labeling was higher in T4 carcinomas relative to lower stage tumors (P = 0.04). High FId(CA9) correlated with the worst disease-free survival rates (P = 0.04). Colocalization between IdUrd labeling and CA9 expression was observed in head and neck squamous cell carcinomas, suggesting the presence of a population of tumor cells under intermediate hypoxic conditions which still has proliferative capacity. The size of this subpopulation may be indicative of tumor aggressiveness and is associated with the worst disease-free survival rates.

INTRODUCTION: Characterisation of glioblastoma (GBM) metabolism is important clinically. The extent to which well-characterised cell lines represent tumour metabolism is poorly described. This study aimed to compare metabolic biomarker expression patterns between a commonly used cell line (U87-MG) and primary GBM cells, in two and three dimensional culture systems. METHOD: U87-MG and primary GBM cells were cultured in serum containing media. Multicellular tumour spheroids were produced by culturing cells in hanging droplet media. Immunofluorescence and fluorescence intensity quantification were used to assess the expression patterns of the following biomarkers: carbonic anhydrase 9 (CA9), glucose transporter 1 (GLUT1), monocarboxylate transporter 4 (MCT4) and glial fibrillary acidic protein (GFAP, control). Independent t-tests (t-t) were used to compare groups. RESULTS: In monolayer culture, CA9 expression was significantly greater in the U87-MG cell line (t-t, t = 3.417, p = 0.012), though GLUT1 (t-t, t = 1.046, p = 0.354) and MCT4 (t-t, t = 0.956, p = 0.393) were similarly expressed. The cell line was also found to be GFAP negative in contrast to primary cultures (t-t, t = 4.323, p = 0.012). In U87-MG spheroids, MCT4 expression was relatively greater in the outermost regions (t-t, t = 2.830, p = 0.047), though CA9 and GLUT1 expression was largely homogeneous. In contrast, primary GBM spheroids displayed gradients of increasing expression towards the outer regions, for all metabolic biomarkers. CONCLUSION: There were differences in metabolic biomarker expression between the cell line and primary GBM cells, in both culture systems. Cell lines should be used with caution to model GBM metabolism.

Stromal cell-derived factor 1α (SDF1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) have been reported to form an important chemokine signaling pathway. Our previous study reported that SDF1α from tumor stromal cells may stimulate the proliferation of gastric cancer (GC) cells through the CXCR4 axis in a hypoxic microenvironment. However, a limited number of studies have addressed the clinicopathological significance of the expression of SDF1α and CXCR4 in GC, particularly at hypoxic regions. Immunohistochemistry was used to investigate the expression levels of SDF1α, CXCR4 and the hypoxic marker carbonic anhydrase 9 (CA9) in 185 patients with stage II and III GC. The results demonstrated that CA9 was expressed on cancer and stromal cells in hypoxic lesions, CXCR4 was mainly expressed in cancer cells, and SDFα was mainly expressed in stromal cells. CXCR4 expression in cancer cells and SDFα expression in stromal cells were associated with the hypoxic regions with CA9 expression. The CA9 and CXCR4 expression in the cancer cells, and the SDF1α expression in the stromal cells (CA9/CXCR4/SDF1α) was significantly associated with macroscopic type 4 tumor (P=0.012) and the pattern of tumor infiltration into the surrounding tissue (P<0.001). The prognosis of the all CA9/CXCR4/SDF1α-positive patients was significantly poorer compared with that of patients with CA9-, CXCR4- or SDF1α-negative GC at Stage III (P=0.041). These results indicated that hypoxia may upregulate SDFα production in stromal cells and CXCR4 expression in cancer cells. The SDF1α/CXCR4 axis may serve an important role in the progression of GC.