Carbonic Anhydrase 9 Expression Research Articles

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

BackgroundPDAC cells upregulate carbonic anhydrase 9 (CA9) expression in order to survive in hypoxic tumor environments, which plays a key role in tumor progression. However, the relationship between CA9 expression and preoperative treatment has not been clarified. We evaluated the clinical impact of CA9 expression on the efficacy of neoadjuvant chemoradiotherapy (NACRT) in pancreatic ductal adenocarcinoma (PDAC). MethodsWe investigated CA9 expression in 273 surgical specimens and 20 serum samples obtained from patients with PDAC and evaluated their clinical outcomes. We analyzed the function of CA9 using human pancreatic cancer cell lines. ResultsCA9 was positively expressed in 36.2 % of patients who underwent NACRT, which was significantly lower than those who underwent upfront surgery (US) (58.9 %, p < 0.001). Interestingly, patients who were CA9-positive in the US group had a significantly poorer prognosis than that of those in the NACRT group (median survival time [MST], 21.5 months vs. 49.2 months, p < 0.001), while there was no significant difference between patients who were CA9-negative in the US and NACRT groups (MST, 45.8 months vs. 46.3 months, p = 0.357). Moreover, serum CA9 levels tended to correlate positively with CA9 expression in cancer tissues. In-vitro experiments demonstrated that CA9 expression was reduced after treatments with radiation and chemoradiation therapy (RT/CRT), and that CA9 knockdown suppressed the impact of RT/CRT on cancer cell proliferation. ConclusionsCA9 may act as a target molecule for RT/CRT, highlighting its clinical importance as a valuable biomarker for more stringent indications for NACRT.

Epigenetic Regulation of Carbonic Anhydrase 9 Expression by Nitric Oxide in Human Small Airway Epithelial Cells.

DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.

Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis.

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a "cold" tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. Invivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as "cold."

IGFL2-AS1-induced suppression of HIF-1α degradation promotes cell proliferation and invasion in colorectal cancer by upregulating CA9.

The lncRNA IGFL2-AS1 is a known cancer-promoting factor in colorectal cancer (CRC); nonetheless, the mechanism of its carcinogenic effects has not yet been elucidated. This study elaborated on the role and underlying molecular mechanism of IGFL2-AS1 in promoting CRC cell functions. IGLF2-AS1 expression levels in CRC tissue/normal tissue and CRC cell line/normal colon epithelial cell line were detected by quantitative real-time polymerase chain reaction. Cell counting kit-8, colony formation assay, and EdU assay were performed to assess the effect of IGFL2-AS1 knockdown or overexpression on the proliferative capacity of CRC cells. The migration and invasion abilities of LoVo cells were measured using transwell assay. The expression relationship between IGFL2-AS1 and carbonic anhydrase 9 (CA9) and the CA9 expression level in CRC tissues and cells was verified by transcriptome sequencing, western blotting, and immunohistochemical staining. Treatment with MG132 and cycloheximide was utilized to explore the mechanism by which IGFL2-AS1 affects the hypoxia-inducible factor-1α (HIF-1α)/CA9 pathway. A nude mouse xenograft model was constructed to evaluate the effect of IGFL2-AS1 on CRC growth in vivo. We discovered that IGFL2-AS1 was highly upregulated in CRC tumor tissues and cells. IGFL2-AS1 can functionally promote CRC cell proliferation, migration, and invasion in vitro and accelerate CRC occurrence in vivo. Mechanistic studies demonstrated that IGFL2-AS1 upregulated the CA9 level by affecting the degradation pathway of HIF-1α, which elucidates its pro-proliferative effect in CRC. The lncRNA IGFL2-AS1 mediated the inhibition of HIF-1α degradation in CRC and increased CA9 expression, thereby promoting CRC progression. Our findings suggested that IGFL2-AS1 is expected to be a promising new diagnostic marker and therapeutic target for CRC.

Gene expression of carbonic anhydrase 9 (CA9) in de novo acute leukemia as a predictive marker for prognosis

Carbonic anhydrase 9 (CA9) is a marker for decreased O2 concentration and acidosis, associated with poor prognosis in cancerous patients. The current study suggested that the changes in CA9 gene expression level might be used as a predictive marker to assess early prognosis at the time of detection of de novo leukemia, and then monitor tumor progress during treatment. This study highlights the level of CA9 gene expression in leukemic patients. A total of 44 cases (acute myeloid leukaemia (AML) group: 23 cases; acute lymphoid leukaemia (ALL) group: 13 cases; control group: 8 healthy volunteers) were selected for this study. The CA9 gene expression was assessed by a real-time PCR with the SYBR green assay. A high level of CA9 gene expression was noticed in AML patients compared to the control group, while the results were not significant in ALL patients. After treatment follow-up, significant differences were observed in CA9 gene expression between a complete response and no response in AML patients. As a result, the CA9 tumor gene could act as a potential early marker for acute leukemia prognosis. A low level of CA9 expression was associated with better clinical outcomes, while a high level was related to a negative prognosis in patients with AML.

Impact of Neoadjuvant Bevacizumab on Neuroradiographic Response and Histological Findings Related to Tumor Stemness and the Hypoxic Tumor Microenvironment in Glioblastoma: Paired Comparison Between Newly Diagnosed and Recurrent Glioblastomas.

BackgroundPreviously, we reported that bevacizumab (Bev) produces histological and neuroradiographic alterations including changes in tumor oxygenation, induction of an immunosupportive tumor microenvironment, and inhibition of stemness. To confirm how those effects vary during Bev therapy, paired samples from the same patients with newly diagnosed glioblastoma (GBM) who received preoperative neoadjuvant Bev (neoBev) were investigated with immunohistochemistry before and after recurrence.MethodsEighteen samples from nine patients with newly diagnosed GBM who received preoperative neoBev followed by surgery and chemoradiotherapy and then autopsy or salvage surgery after recurrence were investigated. The expression of carbonic anhydrase 9 (CA9), hypoxia-inducible factor-1 alpha (HIF-1α), nestin, and Forkhead box M1 (FOXM1) was evaluated with immunohistochemistry.For comparison between neoBev and recurrent tumors, we divided the present cohort into two groups based on neuroradiographic response: good and poor responders (GR and PR, respectively) to Bev were defined by the tumor regression rate on T1-weighted images with gadolinium enhancement (T1Gd) and fluid-attenuated inversion recovery images. Patterns of recurrence after Bev therapy were classified as cT1 flare-up and T2-diffuse/T2-circumscribed. Furthermore, we explored the possibility of utilizing FOXM1 as a biomarker of survival in this cohort.ResultsA characteristic “pseudo-papillary”-like structure containing round-shaped tumor cells clustered adjacent to blood vessels surrounded by spindle-shaped tumor cells was seen only in recurrent tumors. Tumor cells at the outer part of the “pseudo-papillary” structure were CA9-positive (CA9+)/HIF-1α+, whereas cells at the inner part of this structure were CA9−/HIF-1α+ and nestin+/FOXM1+. CA9 and HIF-1α expression was lower in T1Gd-GR and decreased in the “T2-circumscribed/T2-diffuse” pattern compared with the “T1 flare-up” pattern, suggesting that tumor oxygenation was frequently observed in T1Gd-GR in initial tumors and in the “T2-circumscribed/T2-diffuse” pattern in recurrent tumors. FOXM1 low-expression tumors tended to have a better prognosis than that of FOXM1 high-expression tumors.ConclusionA “pseudo-papillary” structure was seen in recurrent GBM after anti-vascular endothelial growth factor therapy. Bev may contribute to tumor oxygenation, leading to inhibition of stemness and correlation with a neuroimaging response during Bev therapy. FOXM1 may play a role as a biomarker of survival during Bev therapy.

Abstract 1040: Blocking hypoxia-induced carbonic anhydrases 9 enhances sensibility to cisplatin of head and neck squamous carcinoma

Abstract The head and neck squamous carcinomas (HNSSCs) are the sixth most common cancer worldwide. Given the heterogeneous nature of HNSCC, this carcinoma results in higher resistance to chemoradiotherapy. Recent evidence highlight the importance of the hypoxic microenvironment in contributing to cisplatin resistance. Therefore, the aim of this study was to investigate the role played by hypoxia-induced protein carbonic anhydrase 9 (CA9) in supporting HNSCC cisplatin resistance. Furthermore, we purpose to investigate the ability of CA9 inhibitor, SLC-0111, a small molecule already used in Phase I clinical trial, to sensitize HNSSC cells to chemotherapy in vitro and in vivo. We analyzed the expression of CA9 in a public data set of 103 HNSCC samples and 22 oral cavity normal tissues and in two HNSSC cell lines (FaDu; SCC-011). HNSCC cells grown in 2D and 3D models under hypoxic conditions (1% O2) showed increased levels of CA9 and greater resistance to cisplatin than cells grown under normoxic conditions. The addition of CA9 inhibitor SLC-0111 to cisplatin sensitized cells to the chemotherapeutic agent and caused a reduction of colony number, spheroid formation, tumor cell, and spheroid migration and invasiveness. Furthermore, the combination therapy hampered activation of STAT3, AKT, ERK, and EMT program whereas it induced apoptosis. Notably, the combined treatment caused inhibition of tumor growth and induction of apoptosis in HNSCC subcutaneous xenografts, as assessed by high-frequency ultrasonography and fluorescent molecular tomography (FMT) with NIR-Annexin V, respectively, at a higher extent than single agents. In addition, FMT with NIR-Prosense revealed a stronger reduction of lymph nodes metastases when mice bearing orthotopic xenografts were treated with cisplatin plus SLC-0111 with respect to a single treatment. Our findings taken together demonstrate the crucial role played by CA9 in promoting cisplatin resistance in HNSCC and the efficacy of SLC-0111 to sensitize tumor cells and xenografts to chemotherapy. Citation Format: Annachiara Sarnella, Ylenia Ferrara, Luigi Auletta, Sandra Albanese, Vincenzo Alterio, Jean-Yves Winum, Claudiu T Supuran, Laura Cerchia, Giuseppina De Simone, Antonella Zannetti. Blocking hypoxia-induced carbonic anhydrases 9 enhances sensibility to cisplatin of head and neck squamous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1040.

1352-P: Differential Metabolomics and Lipidomics Profiles in Low-Birth-Weight Men Unmasked by Four-Weeks High-Carbohydrate Overfeeding

Background: We hypothesized that low birth weight (LBW) subjects with a known increased risk of type 2 diabetes exhibit increased sensitivity towards the deleterious metabolic effects of 4-weeks high carbohydrate overfeeding (HCOF) compared with normal birth weight (NBW) controls. Methods: Untargeted serum metabolomics and lipidomics were measured before and after 4-weeks HCOF (+25% energy) in 26 non-obese men with LBW (2789±174 g) and 21 normal birth weight (NBW) men/controls (3804±172 g) matched for BMI and age. Data were analyzed using a combination of advanced statistical and bioinformatical tools. Results: Among 65 identified metabolites, 8 metabolites were affected differentially by HCOF between the two groups (p&amp;lt;0.05) . In response to HCOF, ingenuity pathway analysis (IPA) revealed increased accumulation and peroxidation of lipids in LBW subjects. In contrast, NBW controls increased albumin synthesis after HCOF. Changes in upstream regulators such as nicotinamide nucleotide transhydrogenase (NNT) , uncoupling protein 1 (UCP1) , and carbonic anhydrase 9 (CA9) were observed in both groups in response to HCOF. NBW controls displayed increased NNT and UCP1 activation, whereas LBW subjects responded with paradoxically decreased NNT and UCP1 activation. CA9 expression was decreased in both groups. Network correlation analysis revealed an upregulation of fatty acids in LBW subjects, contrasting a downregulation among NBW controls. Among 279 lipids identified, the LBW subjects exhibited a significantly higher number of lipids to be increased in response to HCOF (n=26) compared to NBW subjects (n=6) (PFisher=0.0004) , and IPA analysis revealed an activation of the PPARG pathway in NBW as opposed to a paradoxical inhibition in LBW subjects. Conclusion: The results document severe differential perturbations of lipid metabolism in LBW men exposed to HCOF. Public health initiatives promoting low carbohydrate diets in LBW subjects are warranted. Disclosure S.O.Villumsen: None. A.A.Vaag: Stock/Shareholder; AstraZeneca. L.Justesen: None. L.Elingaard-larsen: None. A.B.Thuesen: None. M.Kim: None. C.Legido-quigley: None. E.R.Danielsen: None. T.Hansen: None. C.Brøns: Stock/Shareholder; Novo Nordisk.

The effect of pre-analytical variables on downstream application and data analysis of human endometrial biopsies.

STUDY QUESTIONWhat are the effects of pre-analytical variables on the downstream analysis of patient-derived endometrial biopsies?SUMMARY ANSWERThere are distinct differences in the protein levels of the master regulator of oxygen homeostasis, hypoxia-inducible factor-1-alpha (HIF1α), and the protein and mRNA levels of three related genes, carbonic anhydrase 9 (CA9), vascular endothelial growth factor A (VEGFA) and progesterone receptor (PR) in human endometrial biopsies, depending on the pre-analytical variables: disease status (cancer vs benign), timing of biopsy (pre- vs post-hysterectomy) and type of biopsy (pipelle vs full-thickness).WHAT IS KNOWN ALREADYPatient-derived biopsies are vital to endometrial research, but pre-analytical variables relating to their collection may affect downstream analysis, as is evident in other tissues.STUDY DESIGN, SIZE, DURATIONA prospective observational study including patients undergoing hysterectomy for endometrial cancer (EC) or benign indications was conducted at a large tertiary gynaecological unit in the UK. Endometrial biopsies were obtained at different time points (pre- or post-hysterectomy) using either a pipelle endometrial sampler or as a full-thickness wedge biopsy.PARTICIPANTS/MATERIALS, SETTING, METHODSThe changes in HIF1α, CA9, VEGFA and PR protein levels were measured by semi-quantitative analysis of immunostaining, and the expression levels of three genes (CA9, VEGFA and PR) were investigated by quantitative real-time PCR, in endometrial biopsies from 43 patients undergoing hysterectomy for EC (n = 22) or benign gynaecological indications (n = 21).MAIN RESULTS AND THE ROLE OF CHANCEAn increase in HIF1α immunostaining was observed in EC versus benign endometrium (functionalis glands) obtained pre-hysterectomy (P < 0.001). An increase in CA9 immunostaining was observed in EC versus benign endometrial functionalis glands at both pre- and post-hysterectomy time points (P = 0.03 and P = 0.003, respectively). Compared with benign endometrial pipelle samples, EC samples demonstrated increased mRNA expression of CA9 (pre-hysterectomy P < 0.001, post-hysterectomy P = 0.008) and VEGFA (pre-hysterectomy P = 0.004, post-hysterectomy P = 0.002). In benign uteri, HIF1α immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.009), VEGFA immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.01) and VEGFA mRNA levels (P = 0.008) were increased in matched post-hysterectomy versus pre-hysterectomy samples. Similarly, in EC, an increase in VEGFA immunoscores (epithelial and stromal) and VEGFA mRNA expression was observed in the matched post-hysterectomy versus pre-hysterectomy biopsies (P = 0.008, P = 0.004 and P = 0.018, respectively). Full-thickness benign post-hysterectomy endometrial biopsies displayed increased VEGFA (P = 0.011) and PR (P = 0.006) mRNA expression compared with time-matched pipelle biopsies.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis descriptive study explores the effect of pre-analytical variables on the expression of four proteins and three hypoxia-related genes in a limited number of endometrial biopsies from patients with EC and benign controls. Due to the small number, it was not possible to investigate other potential variables such as menstrual cycle phase, region-specific differences within the endometrium, grade and stage of cancer, and surgical technicalities.WIDER IMPLICATIONS OF THE FINDINGSCareful consideration of the effects of these pre-analytical variables is essential when interpreting data relating to human endometrial biopsies. A standardized approach to endometrial tissue collection is essential to ensure accurate and clinically transferrable data.STUDY FUNDING/COMPETING INTEREST(S)The authors have no conflicts of interest to declare. The work included in this manuscript was funded by Wellbeing of Women project grants RG1073 and RG2137 (D.K.H.), Wellbeing of Women Entry-Level Scholarship ELS706 and Medical Research Council MR/V007238/1 (A.M./D.K.H.), Liverpool Women’s Hospital Cancer Charity (M.A.) and University of Liverpool (L.B., L.R. and E.N.).

Immunohistochemical expression of carbonic anhydrase 9, glucose transporter 1, and paired box 8 in von Hippel-Lindau disease–related lesions

von Hippel-Lindau (VHL) disease occurs secondary to pathogenic alterations of the VHL tumor suppressor gene, manifesting with cysts and tumors in multiple organ systems. VHL protein (pVHL) is a known downregulator of hypoxia inducible factor-1a (HIF1a). Loss of function of pVHL is associated with upregulation of the HIF1a pathway including carbonic anhydrase 9 (CA9)and glucose transporter 1 (GLUT1). Paired box 8 (PAX8) is an important transcription factor regulator of mesonephric development. We investigated the role of immunohistochemistry in the assessment of CA9, GLUT1, and PAX8 expression in VHL disease-related lesions. Clinicopathologic information and archived pathology material from 5 patients with VHL disease were reviewed and evaluated for expression ofCA9, GLUT1, and PAX8. The spectrum of VHL disease-related lesions includedhemangioblastoma, endolymphatic sac tumor, pulmonary microcysts, pheochromocytoma, pancreatic neuroendocrine tumor and serous cystadenoma, renal cysts, renal cell carcinoma, and epididymal papillary cystadenoma. CA9 was expressed in all lesions and exhibited diffuse positivity (15/15 lesions, 100%; 5/5 patients), while GLUT1 expression was focal/weak or absent in some instances (strong positive: 12/15 lesions, 80%; 5/5 patients). PAX8 was expressed only in renal and epididymal lesions. CA9 and GLUT1 are consistently overexpressed in VHL disease-related lesions, reflecting upregulation of the HIF1a pathway. PAX8 is only expressed in genitourinary lesions, mirroring organ-specific differentiation. A combination of CA9 and GLUT1 immunostains is useful in screening lesions of patients with VHL spectrum manifestations, which may be targeted by the recently Food and Drug Administration-approved HIF-2a inhibitors.

CA9-Related Acidic Microenvironment Mediates CD8+ T Cell Related Immunosuppression in Pancreatic Cancer.

PurposeThis study aims to integrate pancreatic cancer TCGA, GEO, and single-cell RNA-sequencing (scRNA-seq) datasets, and explore the potential prognostic markers and underlying mechanisms of the immune microenvironment of pancreatic cancer through bioinformatics methods, in vitro and in vivo assays.MethodsExpression data and clinicopathological data of pancreatic cancer TCGA, GEO (GSE131050), single cell sequencing (PAAD_CRA001160) dataset were downloaded. We used R/Bioconductor edgeR for differential expression analysis. ClusterProfiler was utilized to perform GO enrichment analysis on differentially expressed genes. The online software CIBERSORT was used to reanalyze the mRNA expression data of pancreatic cancer. CellRanger, RunPCA, FindNeighbors, FindClusters, RunTSNE and RunUMAP were used to perform preprocessing, cell clustering and expression profile analysis on single-cell sequencing data sets. We analyzed intracellular pH with or without CA9 inhibitor SLC-0111. Indirect co-culture model of human pancreatic cancer cell lines and healthy individual-derived PBMCs were used to determine the effect of CA9-related Acidic Microenvironment on CD8+ T cells.ResultsThe CIBERSORT analysis of TCGA pancreatic cancer transcriptome sequencing data showed that among the 22 immune microenvironment components, CD8+ T cell infiltration was significantly correlated with the prognosis of pancreatic cancer patients. The differential expression analysis of the TCGA data grouped by the level of CD8+ T cell infiltration indicates that the expression of carbonic anhydrase 9 (CA9) is the most significant, and the survival analysis suggests that CA9 is associated with the overall survival of pancreatic cancer. TCGA data and GEO data set GSE131050 expression correlation analysis suggests that CA9 and CD8 expression are closely related. Pancreatic cancer single-cell sequencing data set PAAD_CRA001160 analysis results show that CA9 is mainly expressed in pancreatic cancer cell clusters, and the expression of the cancer cell subgroup CA9 in the single-cell data set is correlated with CD8+ T cell infiltration.ConclusionPancreatic cancer cells may inhibit the infiltration of CD8+ T cells through CA9. Further exploration of its related mechanisms can be used to explore the immune escape pathway of pancreatic cancer and provides new perspectives immune targeted therapy.

Mitochondrial Ndufa4l2 Enhances Deposition of Lipids and Expression of Ca9 in the TRACK Model of Early Clear Cell Renal Cell Carcinoma.

Mitochondrial dysfunction and aberrant glycolysis are hallmarks of human clear cell renal cell carcinoma (ccRCC). Whereas glycolysis is thoroughly studied, little is known about the mitochondrial contribution to the pathology of ccRCC. Mitochondrial Ndufa4l2 is predictive of poor survival of ccRCC patients, and in kidney cancer cell lines the protein supports proliferation and colony formation. Its role in ccRCC, however, remains enigmatic. We utilized our established ccRCC model, termed Transgenic Cancer of the Kidney (TRACK), to generate a novel genetically engineered mouse model in which dox-regulated expression of an shRNA decreases Ndufa4l2 levels specifically in the renal proximal tubules (PT). This targeted knockdown of Ndufa4l2 reduced the accumulation of neutral renal lipid and was associated with decreased levels of the ccRCC markers carbonic anhydrase 9 (CA9) and Enolase 1 (ENO1). These findings suggest a link between mitochondrial dysregulation (i.e. high levels of Ndufa4l2), lipid accumulation, and the expression of ccRCC markers ENO1 and CA9, and demonstrate that lipid accumulation and ccRCC development can potentially be attenuated by inhibiting Ndufa4l2.

Impact of p53, HIF1a, Ki-67, CA-9, and GLUT1 Expression on Treatment Outcomes in Locally Advanced Cervical Cancer Patients Treated With Definitive Chemoradiation Therapy

The objective of this study was to assess the association between pretreatment p53, hypoxia inducible factor 1a (HIF1a), Ki-67, carbonic anhydrase-9 (CA-9), and glucose transporter 1 (GLUT1) expression in locally advanced cervical cancer patients treated definitively with concurrent chemoradiation therapy (CRT) and treatment outcomes including overall survival (OS), progression-free survival (PFS), local-regional control (LC), and distant metastases-free survival (DMFS). Twenty-eight patients treated definitively and consecutively for cervical cancer with CRT had p53, HIF1a, Ki-67, CA-9, and GLUT1 protein expression assessed and scored semiquantitatively by 3 pathologists, blinded to the treatment outcomes. Outcomes were stratified by p53 (H-score: <15 vs. ≥15), HIF1a (H-score: <95 vs. ≥95), Ki-67 (labeling index <41% vs. ≥41%), CA-9 (H-score: <15 vs. ≥15), and GLUT1 (H-score: <175 vs. ≥175) expression. OS, PFS, LC, and DMFS rates were calculated using the Kaplan-Meier method, and differences between groups were evaluated by the log-rank test. Notable clinical characteristics of the cohort included median age of 51 years (range: 32 to 74 y), FIGO stage IIB disease (57.2%), clinical node-negative disease (64.3%), squamous cell carcinoma (89.3%), and adenocarcinoma (10.7%). Treatment outcomes included 5-year OS (57.2%), PFS (48.1%), LC (72.1%), and DMFS (62.9%). For HIF1a H-score <95 and ≥95, the 5-year OS (52.0% and 68.4%, P=0.58), PFS (53.0% and 40.9%, P=0.75), LC (71.6% and 68.2%, P=0.92), and DMFS (59.7% and 52.0%, P=0.91) were not significantly different. For Ki-67 labeling index <41% and ≥41%, the 5-year OS (44.9% and 66.6%, P=0.35), PFS (38.9% and 55.4%, P=0.53), LC (57.7% and 85.7%, P=0.22), and DMFS (67.3% and 61.0%, P=0.94) were not significantly different. For CA-9 H-score <15 and ≥15, the 5-year OS (54.4% and 66.7%, P=0.39), PFS (57.3% and 40.0%, P=0.87), LC (70.0% and 70.0%, P=0.95), and DMFS (70.0% and 46.7%, P=0.94) were not significantly different. For GLUT1 H-score <175 and ≥175, the 5-year OS (43.6% and 43.6%, P=0.32), PFS (55.6% and 49.5%, P=0.72), LC (72.9% and 71.5%, P=0.97), and DMFS (62.5% and 59.6%, P=0.76) were not significantly different. For p53, H-score <15 and ≥15, the 5-year OS (62% and 53%), PFS (63% and 30.3%), LC (87.5% and 52%), and DMFS (79.6% and 41.6%). In this study population, HIF1a, Ki-67, CA-9, and GLUT1 expression did not predict treatment response or outcomes in locally advanced cervical cancer patients treated definitively with CRT. There was a nonstatistically significant trend towards worse outcomes with p53 expression.

Characterization of CD147, CA9, and CD70 as Tumor-Specific Markers on Extracellular Vesicles in Clear Cell Renal Cell Carcinoma.

Extracellular vesicles (EVs) are secreted by healthy and tumor cells and are involved in cell–cell communication. Tumor-released EVs could represent a new class of biomarkers from liquid biopsies. The aim of this study was to identify tumor-specific EV markers in clear cell renal carcinoma (ccRCC) using cell lines and patient-derived tissue samples. EVs from ccRCC cell lines (786-O, RCC53, Caki1, and Caki2) and patient tissues were isolated via ultracentrifugation. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting using exosome and putative tumor markers (epithelial cell adhesion molecule (EpCAM), carbonic anhydrase 9 (CA9), CD70, CD147). The tumor markers were verified using immunohistochemistry. CA9 was expressed in Caki2 cells and EVs, and CD147 was found in the cells and EVs of all tested ccRCC cell lines. In tumor tissues, we found an increased expression of CA9, CD70, and CD147 were increased in cell lysates and EV fractions compared to normal tissues. In contrast, EpCAM was heterogeneously expressed in tumor samples and positive in normal tissue. To conclude, we developed an effective technique to isolate EVs directly from human tissue samples with high purity and high concentration. In contrast to EpCAM, CA9, CD70, and CD147 could represent promising markers to identify tumor-specific EVs in ccRCC.

SDF1α/CXCR4 axis may be associated with the malignant progression of gastric cancer in the hypoxic tumor microenvironment.

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.

Correlation of carbonic anhydrase 9 (CA9) with pathological T-stage and prognosis in patients with oral tongue squamous cell carcinoma.

BackgroundWe explored the mechanisms underlying tumorigenesis in oral tongue squamous cell carcinoma (OTSCC) with the goal of uncovering prognostic molecular biomarkers.MethodsAn mRNA sequencing dataset was obtained from The Cancer Genome Atlas (TCGA) database, and differentially expressed genes (DEGs) were selected using R language software packages. Functional enrichment analysis was conducted with DAVID software and protein-protein interaction (PPI) networks were constructed using the STRING database. The relationship between hub genes and overall survival (OS) was evaluated by Kaplan-Meier analysis and Cox proportional hazard regression models. Expression of the candidate gene, carbonic anhydrase 9 (CA9), was verified by real-time RT-PCR, western blotting, and immunohistochemistry.ResultsDEGs (n=581) were obtained from 11 OTSCC samples and corresponding adjacent non-tumor tissues. Gene ontology (GO) analysis revealed that most DEGs were implicated in anterior/posterior pattern specification, embryonic skeletal system morphogenesis, and multicellular organism development, and pathway analysis suggested that DEGs were associated with neuroactive ligand-receptor interaction, calcium signaling pathway and transcriptional misregulation in the cancer. A PPI network consisting of 301 nodes and 2011 edges was constructed and 71 hub genes, with high degrees of connectivity in the network, were identified. Kaplan-Meier analysis of the hub genes indicated that high expression of CA9, LHX1, and KISS1R and low expression of CCKAR were associated with poor OS in OTSCC; however, only CA9 was a significant prognostic factor influencing survival in OTSCC on multivariate analysis. High expression of CA9 was associated with poor pathological T-stage. CA9 tumor specificity was confirmed using the Gene Expression Omnibus (GEO) database and further molecular tests.ConclusionsWe identified key DEGs that may assist in the molecular understanding of OTSCC. CA9 warrants further exploration as potential prognostic biomarker and therapeutic target in OTSCC.

Carbonic anhydrase IX (CA9) expression in multiple renal epithelial tumour subtypes.

Renal epithelial neoplasms (RENs) can be difficult to subclassify, owing to overlapping morphological features. Carbonic anhydrase 9 (CA9) is a common biomarker for clear cell renal cell carcinoma (CCRCC); however, the sensitivity and specificity across REN subtypes are less clear. The aim of this study was to investigate CA9 expression in RENs, especially those in the differential diagnosis with CCRCC and less common entities, to determine its reliability as a diagnostic biomarker. CA9 immunostaining was performed on 262 RENs, including 119 CCRCCs and 143 non-CCRCC. Immunostaining was evaluated as negative (0%), rare (1+, 1-10%), focal (2+, 11-50%), or diffuse (3+, >50%). CCRCCs were 3+ CA9-positive in 93% of cases; 4% were CA9-negative. Sixty-seven percent of papillary renal cell carcinomas (RCCs) were 1+/2+ CA9-positive, whereas 33% were CA9-negative. Chromophobe RCCs were nearly always CA9-negative (93%), with 7% showing rare cell reactivity. Clear cell tubulopapillary RCCs (CCTPRCCs) were consistently 3+ CA9-positive, but with a cup-like staining pattern. Fifty-three percent of Xp11.2 RCCs were CA9-negative; however, 6% were 3+ CA9-positive and 12% were 2+ CA9-positive. Two of eight fumarate hydratase-deficient RCCs were 3+ CA9-positive. A small subset of the remaining RCCs showed rare to focal CA9 expression. All oncocytomas and eosinophilic solid and cystic RCCs were CA9-negative. Overall, diffuse CA9 expression was identified in nearly all CCRCCs and in all CCTPRCCs (high sensitivity); however, CA9 was not entirely specific. At least focal CA9 expression can been seen in a subset of many RCCs, and such findings should be taken into consideration with other morphological, immunophenotypic and clinical findings.

Programmed death ligand-1 (PD-L1) expression in meningioma; prognostic significance and its association with hypoxia and NFKB2 expression

Management of clinically aggressive meningiomas is a considerable challenge. PD-L1 induced immune suppression has increasingly gained attention in clinical management of cancer; however, to date, the clinical significance and regulatory mechanisms of PD-L1 in meningioma is not yet fully characterized. We sought to characterize PD-L1 expression in meningioma and elucidate its regulatory mechanisms. Immunohistochemical staining of PD-L1 expression in meningiomas showed 43% positivity in both tumor and immune cells and we observed intra and inter tumoral heterogeneity. Univariate and multivariate analyses confirmed that PD-L1 protein expression is an independent prognostic marker for worse recurrence free survival in meningioma. Furthermore, our transcriptomic analysis revealed a strong association between PD-L1 expression and that of NFKB2 and carbonic anhydrase 9 (CA9). We also demonstrated that both of these markers, when co-expressed with PD-L1, predict tumor progression. Our studies on several meningioma cell lines cultured in hypoxic conditions validated the association of CA9 and PD-L1 expression. Here we show the clinical significance of PD-L1 in meningioma as a marker that can predict tumor recurrence. We also show an association PD-L1 expression with NFKB2 expression and its induction under hypoxic conditions. These findings may open new avenues of molecular investigation in pathogenesis of meningioma.

Abstract 5806: Hypoxia-related radiomics predict checkpoint blockade immunotherapy response of non-small cell lung cancer patients

Abstract Checkpoint blockade immunotherapy exhibits durable responses in a subset of advanced stage non-small cell lung cancer (NSCLC) patients. However, not all patients respond and markers that predict response to immunotherapy are still an unmet clinical need. In this study, pre-treatment quantitative image-based biomarkers (radiomics) were utilized to predict survival risk among NSCLC patients treated with immunotherapy. Study cohorts were compromised of NSCLC patients that were treated with single or double agent immunotherapy and were divided into three independent groups from two different hospitals for Training (N = 180), test (N = 90) and validation (N = 62). Overall survival (OS) and progression-free survival (PFS) were used as endpoints. The most prognostic radiomic features and clinical covariates were used as inputs to a Classification and Regression Tree (CART) machine learning algorithm to stratify patients into survival risk-groups utilizing the training cohort. The risk groups identified were then tested and validated in the test and validation cohorts. Additionally, four independent non-immunotherapy treated NSCLC cohorts (total N = 446) were utilized to further investigate the prognostic value of the derived radiomics signature. The biological underpinnings of the most informative radiomics were assessed using gene expression data from a radiogenomics dataset and an immunohistochemistry (IHC) data.The final parsimonious model was able to stratify patients into four risk groups (from low-risk to very-high-risk) and the models were successfully tested and validated in independent cohorts. Specifically, the very-high-risk group was found to be associated with extremely poor OS outcomes (0% 3-year OS; hazard ratio [HR] = 5.35, 95% confidence interval [CI]: 2.14 - 13.36) compared to the low-risk group (38.9% 3-year OS; HR = 1.00). When PFS was assessed, similar outcomes were observed (0% vs. 29.8% 3-year PFS). A radiomic feature that quantifies the heterogeneity of tumor structural phenotype (GLCM inverse difference) was highly associated with OS in all three immunotherapy treated cohorts. Utilizing gene expression data, GLCM inverse difference was found to be positively associated with expression of carbonic anhydrase-9 (CA-IX), a surrogate for tumor hypoxia. This was validated by IHC, and further analysis showed that it was also associated with OS in the four other independent non-immunotherapy treated NSCLC cohorts.In this study, we utilized non-invasive image-based features and standard-of-care clinical covariates to identify novel risk groups that can predict OS and PFS among NSCLC patients treated with IO. These models have important translational associations as a potential risk score that could be used to assist decision making by identifying potentially vulnerable patients in the setting of immunotherapy. Citation Format: Ilke Tunali, Yan Tan, Jhanelle E. Gray, Evangelia Katsoulakis, Steven A. Eschrich, James Saller, Theresa Boyle, Jin Qi, Albert Guvenis, Robert J. Gillies, Matthew B. Schabath. Hypoxia-related radiomics predict checkpoint blockade immunotherapy response of non-small cell lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5806.

Carbonic anhydrase 9 (CA9) expression in non-small-cell lung cancer: correlation with regulatory FOXP3+T-cell tumour stroma infiltration

BackgroundLow pH suppresses the proliferation and cytotoxic activity of CD8+ cytotoxic and natural killer lymphocytes. The hypoxia-regulated transmembrane protein, carbonic anhydrase CA9, converts carbon dioxide produced by the Krebs cycle to bicarbonate and protons that acidify the extracellular milieu. We examined whether CA9 is also involved in intratumoural immunosuppression pathways.MethodsA series of 98 tissue samples of primary non-small-cell lung carcinomas (NSCLC) from patients treated with surgery were analysed for the expression of CA9 and programmed-death ligand PD-L1 by cancer cells, and of FOXP3 by tumour-infiltrating lymphocytes (TILs).ResultsThere was no direct association of CA9 with PD-L1 expression or the density of TILs in the tumour stroma, but CA9 was directly related to the extent of FOXP3+ TIL density (p = 0.008). Double-stratification survival analysis showed that patients with high CA9 expression and low TIL score had significantly poorer survival compared with all other groups (p < 0.04). In a multivariate analysis stage (p < 0.0001, HR 1.95, 95% CI: 1.3–2.7), TIL score (p = 0.05, HR 0.55, 95% CI: 0.2–1.0) was an independent prognostic variable of death events. CA9 expression by cancer cells is associated significantly with FOXP3+ regulatory T-cell abundance in the tumour stroma of NSCLC.ConclusionThe study provides a basis for testing CA9 as a marker of resistance to immune-checkpoint inhibitors and as a therapeutic target to enhance the efficacy of immunotherapy.