Low Carbonic Anhydrase IX Expression Research Articles

A CAIX Dual-Targeting Small-Molecule Probe for Noninvasive Imaging of ccRCC.

Carbonic anhydrase IX (CAIX), a zinc metal transmembrane protein, is highly expressed in 95% of clear cell renal cell carcinomas (ccRCCs). A positron emission tomography (PET) probe designed to target CAIX in nuclear medicine imaging technology can achieve precise positioning, is noninvasive, and can be used to monitor CAIX expression in lesions in real time. In this study, we constructed a novel acetazolamide dual-targeted small-molecule probe [68Ga]Ga-LF-4, which targets CAIX by binding to a specific amino acid sequence. After attenuation correction, the radiolabeling yield reached 66.95 ± 0.57% (n = 5) after 15 min of reaction and the radiochemical purity reached 99% (n = 5). [68Ga]Ga-LF-4 has good in vitro and in vivo stability, and in vivo safety and high affinity for CAIX, with a Kd value of 6.62 nM. Moreover, [68Ga]Ga-LF-4 could be quickly cleared from the blood in vivo. The biodistribution study revealed that the [68Ga]Ga-LF-4 signal was concentrated in the heart, lung, and kidney after administration, which was the same as that observed in the micro-PET/CT study. In a ccRCC patient-derived xenograft (PDX) model, the signal significantly accumulated in the tumor after administration, where it was retained for up to 4 h. After competitive blockade with LF-4, uptake at the tumor site was significantly reduced. The SUVmax of the probe [68Ga]Ga-LF-4 at the ccRCC tumor site was three times greater than that in the PC3 group with low CAIX expression at 30 min (ccRCC vs PC3:1.86 ± 0.03 vs 0.62 ± 0.01, t = 48.2, P < 0.0001). These results indicate that [68Ga]Ga-LF-4 is a novel small-molecule probe that targets CAIX and can be used to image localized and metastatic ccRCC lesions.

Abstract 4004: Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma

Abstract On-target off-tumor toxicity (OTOT) is one of the major hurdles preventing tumor-associated antigen (TAA) targeted therapy from effectively treating patients with solid tumors due to the shared presence of the same TAA on normal tissues. We have previously demonstrated high expression of carbonic anhydrase IX (CAIX) in ccRCC patient samples and low-density expression on healthy bile duct epithelium. In 2006, a clinical trial was stopped due to OTOT bile duct toxicity observed in patients infused with anti-CAIX CAR T cell therapy. To address the issue of OTOT in the bile duct, we performed affinity/avidity fine-tuning of the anti-CAIX CAR G36 to specifically target ccRCC tumor cells with high CAIX expression while sparing cholangiocytes with low CAIX expression. We generated a complementarity-determining region (CDR) single variant mutagenesis library of G36 and displayed the library on yeast for screening. Using flow cytometry, we were able to identify six populations based on antigen-binding affinity: high, med-high, med, med-low, low, and loss of binding. These libraries were sequenced via PacBio, a next generation sequencing (NGS) platform that excels in long-read sequencing, providing a powerful tool to detect single mutations in the scFv. The results were analyzed to identify enriched mutations compared to the wild-type G36, and inspected using in silico modeling and docking. Furthermore, enriched mutants from med-low and low populations were tested in CAR-T format for their cytotoxic capacity on tumor cells and bile duct cholangiocytes, as well as their avidity. We demonstrate that mutations in CDR2 and CDR3 of the heavy chain successfully mitigate OTOT toxicity on cholangiocytes with physiological levels of CAIX expression while maintaining tumor-killing capacity, shedding light on CDR mutagenesis as a useful tool to mitigate OTOT toxicity. Citation Format: Stephen Hsien-Chi Yuan, Yufei Wang, Rabia Abbas, Peterson Mathenge, Charles Chien, Christian D. Coherd, Branson Bajoua, Matthew R. Chang, Nithyassree Murugan, Gabriella M. Kastrunes, Guilin Wang, Song-My Hoang, Wayne A. Marasco. Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4004.

Spatial transcriptomic analysis of tumour with high and low CAIX expression in TNBC tissue samples using GeoMx™ RNA assay.

Prognostic significance and gene signatures associated with carbonic anhydrase IX (CAIX) was investigated in triple negative breast cancer (TNBC) patients. Immunohistochemistry (IHC) for CAIX was performed in tissue microarrays (TMAs) of 136 TNBC patients. In a subset of 52 patients Digital Spatial Profiler (DSP) was performed in tumour (pan-cytokeratin+) and stroma (pan-cytokeratin-). Differentially expressed genes (DEGs) with P&lt;0.05 and and log2 fold change (FC)>(±0.25 and ±0.3, for tumour and stromal compartment, respectively) were identified. Four genes were validated at the protein level. Cytoplasmic CAIX expression was independently associated with poor recurrence free survival in TNBC patients [hazard ratio (HR)=6.59, 95% confidence interval (CI): 1.47-29.58, P=0.014]. DEG analysis identified 4 up-regulated genes (CD68, HIF1A, pan-melanocyte, and VSIR) in the tumour region and 9 down-regulated genes in the stromal region (CD86, CD3E, MS4A1, BCL2, CCL5, NKG7, PTPRC, CD27, and FAS) when low versus high CAIX expression was explored. Employing IHC, high CD68 and HIF-1α was associated with poorer prognosis and high BCL2 and CD3 was associated with good prognosis. DSP technology identified DEGs in TNBC. Selected genes validated by IHC showed involvement of CD3 and BCL2 expression within stroma and HIF-1α, and CD68 expression within tumour. However, further functional analysis is warranted.

Prognostic Significance of Membranous Carbonic Anhydrase IX Expression in Patients with Nonmetastatic Clear Cell Renal Cell Carcinoma of Different Tumor Stages.

Background: There are paradoxical results regarding whether carbonic anhydrase IX (CAIX) is a prognostic biomarker for patients with clear cell renal cell carcinoma (ccRCC). The objective of this study was to evaluate prognostic significance of CAIX in nonmetastatic ccRCC patients of different stages. Materials and Methods: This is a retrospective study on 1263 patients with nonmetastatic ccRCC from January 2005 to June 2018. Patients were stratified into eight subgroups (pT1a, pT1b, pT2a, pT2b, pT3a, pT3b, pT3c, and pT4) according to the 2016 TNM classification system. Immunohistochemical staining of membranous CAIX was quantified. Cancer-specific survival (CSS) rates in patients with high (>85%) and low (<85%) CAIX expressions were compared by Kaplan-Meier curves with log-rank test. Results: There were 220 tumors (17.42%) with low CAIX expression and 1043 tumors (82.58%) with high CAIX expression. The cumulative CSS rates were statistically significant between all patients with low and high CAIX expression (p-value <0.001). In pT2a, pT2b, and pT3a subgroups, the patients with low CAIX expression exhibited markedly decreased cumulative CSS rates compared to patients with high CAIX expression (p-value <0.05). Univariable and multivariable Cox regression analysis showed that CAIX expression was an independent predictor of prognosis in patients with pT2a, pT2b, and pT3a ccRCC (p-value <0.05), rather than in all nonmetastatic patients. Conclusion: CAIX expression is of independent prognostic value for ccRCC patients in pT2a, pT2b, and pT3a stages. CAIX expression combined with tumor stage would further improve risk stratification of nonmetastatic ccRCC patients and provide directions for therapies.

Targeting CAIX with [64Cu]XYIMSR-06 Small Molecular Radiotracer Enables Noninvasive PET Imaging of Malignant Glioma in U87 MG Tumor Cell Xenograft Mice.

Carbonic anhydrase IX (CAIX) plays an important role in glioma cell proliferation, invasion, metastasis, and resistance to radiotherapy and chemotherapy. An effective and noninvasive PET molecular imaging agent targeting CAIX would help its diagnosis and treatment but is not currently available. Recently, a low-molecular-weight (LMW) CAIX targeting agent, [64Cu]XYIMSR-06, was reported to have significantly improved properties for targeting clear cell renal cell carcinoma (ccRCC). We are encouraged to investigate the feasibility of adapting this agent for the diagnosis and treatment of CAIX-overexpressing malignant glioma. In vitro cell uptake and binding affinity assays were used to verify the binding capacity of [64Cu]XYIMSR-06 to U87 MG tumor cells in which CAIX overexpression was confirmed. The U87 MG tumor-bearing mouse (in situ and subcutaneous) model was built, and mice were injected with the radiotracer and/or coinjected with acetazolamide (0.2 g/kg) as a blocking agent for noninvasive micro-PET imaging. Micro-PET imaging was performed at 2, 4, and 8 h postinjection. ROI (region of interest)-based semiquantification was performed in an orthotopic glioma tumor model. Biodistribution throughout each organ was performed at 2, 4, 4 h block, 8, and 24 h postinjection. Hematoxylin and eosin (HE) staining and immunofluorescence or immunohistochemistry (IF/IHC) staining were implemented postimaging to assess the expression of CAIX in tumor organs. In vitro, [64Cu]XYIMSR-06 exhibits greater uptake in glioma cells (high CAIX expression) than in HCT116 cells (low CAIX expression). The binding affinity of [64Cu]XYIMSR-06 to U87 MG cell lines reaches up to 4.22 nM. Both orthotopic and subcutaneous tumors were clearly visualized at 2-8 h postinjection. Biodistribution studies demonstrated a maximum tumor uptake of 3.13% ID/g at 4 h postinjection, and the tumor to brain ratio (T/brain) was 6.51 at 8 h postinjection. The ROI-based T/brain values were 7.03 and 5.46 at 2 and 8 h postinjection, respectively. Histopathological analysis confirmed the overexpression of CAIX in gliomas, and the area of CAIX-positive IF staining is extremely consistent with the morphology on micro-PET imaging. In this study, [64Cu]XYIMSR-06 demonstrated specific accumulation in CAIX-expressing U87 MG glioma tumors, indicating that the radiotracer has the potential for noninvasively monitoring and guiding personalized treatment of malignant glioma and other tumors overexpressing CAIX.

Utility of multiphasic multidetector computed tomography in discriminating between clear cell renal cell carcinomas with high and low carbonic anhydrase-IX expression.

To investigate if multiphasic multidetector computed tomography (MDCT) enhancement profiles can distinguish clear cell renal cell carcinomas (ccRCCs) with high carbonic anhydrase-IX (CA-IX) expression from ccRCCs with low CA-IX expression. With IRB approval for this retrospective study, we derived a cohort of 105 histologically proven ccRCCs with preoperative 4-phase renal mass MDCT from 2001 to 2013. Following manual segmentation, the computer-assisted detection algorithm selected a 0.5-cm-diameter region of maximal attenuation within each lesion in each phase. CA-IX expression level was determined by immunohistochemical staining of tumor specimens. In the high and low CA-IX expression subgroups, the magnitude of enhancement and washout were compared using t tests; the performance of contrast washout in differentiating between subgroups was assessed with logistic regression analysis. ccRCCs with high and low CA-IX expression both exhibited peak enhancement in the corticomedullary phase. ccRCCs with high CA-IX expression demonstrated significantly greater relative nephrographic washout than those with low CA-IX expression (18.4% vs. 7.8%, p=0.03). ccRCCs with high CA-IX expression had greater relative excretory washout than ccRCCs with low CA-IX expression with a trend toward significance (33.4% vs. 25.2%, p=0.05). After controlling for tumor size and stage, for distinguishing ccRCCs with high and low CA-IX expression, relative excretory washout had a sensitivity, negative predictive value, accuracy, and positive predictive value of 99% (65/66), 88% (7/8), 69% (72/105), and 67% (65/97), respectively. Relative nephrographic and excretory washout may have the potential to help distinguish ccRCCs with high and low CA-IX expression, but this requires further validation.

Abstract 3780: On the role of experimental microenvironmental conditions in targeted inhibition of the pH-regulating carbonic anhydrase IX in colorectal carcinoma cells

Abstract Tumor hypoxia, causing metabolic changes towards enhanced glycolysis and lactic acid production, is a common microenvironmental feature of solid tumors, and is recognized as a main mechanism both of therapeutic resistance and metastasis. The hypoxia-inducible factor-1α conducts transcription of a number of genes involved in adaption to the hypoxic condition. Among these is the tumor-specific carbonic anhydrase IX (CAIX), a membrane-bound protein that regulates extracellular (pHe) and intracellular (pHi) pH by catalyzing the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and protons, enabling tumor cell survival in the acidic microenvironment. To improve therapeutic outcome and reduce metastatic risk, targeted inhibition of CAIX is an attractive strategy. Here we report on effects of a CAIX inhibitor in a panel of colorectal carcinoma (CRC) cell lines, giving specific emphasis to the microenvironmental conditions. Five CRC cell lines were incubated for 24 hours under normoxic (21% O2) or hypoxic (0.2% O2) condition, either in the presence (22 mM HCO3-, 5% CO2) or absence (0 mM HCO3-, 0% CO2) of HCO3- in the culture medium. The cells were treated with 4-(3′-(3”,5”-dimethylphenyl)ureido)phenyl sulfamate (termed S4), considered to be a CAIX-specific inhibitor, and clonogenicity, pHe, and pHi (measured as cells labeled with a pH-sensitive dye) were assessed. In CO2/HCO3--buffered medium under normoxia, differential inhibition of clonogenicity following S4 treatment was observed among the five cell lines, correlating with individual CAIX levels: strong suppression in cell lines with high CAIX expression, weaker S4 effect in cell lines with low expression, and no effect in the CAIX-negative cell line. When incubating with this medium under hypoxia, despite significant induction of CAIX, no additional effect of S4 on clonogenicity was observed. Of note, when changing the cell culture medium to CO2/HCO3--free condition, S4 strongly inhibited clonogenicity of hypoxic HT29 cells (high CAIX expression) but not of the normoxic counterpart, while HCT116 cells (low CAIX expression) showed no clonogenic response to S4 with either oxygenation status. In all of the five cell lines, hypoxia caused significant acidification (decrease in pHe) of culture media. In parallel with the clonogenic response, S4 treatment significantly counteracted the hypoxia-induced pHe decline in HT29 cultures incubated in the CO2/HCO3--free condition. pHi was not altered with any of the tested conditions. This set of data demonstrated that the CO2/HCO3- buffering system determined CRC cell response to targeted CAIX inhibition. To enable reliable assessment of experimental S4 effects, strict control of the microenvironmental conditions was required. We believe this information may be of note for future evaluation of this specific therapeutic approach. Citation Format: Helga Helseth Hektoen, Kjersti Flatmark, Kirsti Solberg Landsverk, Marie Gron Saelen, Kathrine Roe, Anne Hansen Ree. On the role of experimental microenvironmental conditions in targeted inhibition of the pH-regulating carbonic anhydrase IX in colorectal carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3780. doi:10.1158/1538-7445.AM2014-3780

Carbonic anhydrase IX (CAIX) is not an independent predictor of outcome in patients with clear cell renal cell carcinoma (ccRCC) after long‐term follow‐up

To evaluate carbonic anhydrase IX (CAIX) expression as an independent prognostic marker for clear cell renal cell carcinoma (ccRCC). With recent smaller studies showing conflicting results, we aimed to update our initial analysis in 2007 with an additional 5-year follow-up. We provided long-term follow-up of the same cohort used in our 2007 study (730 patients with unilateral, sporadic ccRCC treated surgically between 1990 and 1999). Associations of CAIX expression with RCC death and distant metastases were evaluated using Cox proportional hazards regression models. CAIX was expressed in 708 (97.0%) of the specimens; 163 tumours (22.3%) had low (≤85%) expression and 567 (77.7%) high (>85%) expression. There were 483 deaths and 265 RCC-specific deaths. The median follow-up for the 247 patients still under observation was 13.8 years. Univariately, low CAIX expression was associated with an increased risk of RCC death vs high expression (hazard ratio 1.62; P < 0.001). Low CAIX expression was not statistically significantly associated with RCC death or distant metastases after adjusting for nuclear grade or coagulative tumour necrosis. After additional long-term follow-up of our large cohort, our results continue to suggest that CAIX is not an independent prognostic marker for ccRCC.

Carbonic anhydrase IX as a potential biomarker of efficacy in metastatic clear-cell renal cell carcinoma patients receiving sorafenib or placebo: Analysis from the treatment approaches in renal cancer global evaluation trial (TARGET)

ObjectiveRetrospective data analyses have suggested that carbonic anhydrase IX (CAIX) may have a predictive role in patients with metastatic clear cell renal cell carcinoma (ccRCC) receiving high dose interleukin-2 or sorafenib. We examined the predictive value of CAIX in estimating treatment outcome in patients receiving sorafenib vs. placebo as part of the Treatment Approaches in Renal Cancer Global Evaluation Trial (TARGET) study. Materials and methodsParaffin embedded tumor tissues were collected from 133 patients from the TARGET study (n = 903). The percentage of CAIX-positive cells was assessed by a single pathologist. The impact of CAIX expression on progression-free survival (PFS, primary endpoint) and tumor shrinkage (TS, secondary endpoint) was analyzed. ResultsClinical characteristics were similarly distributed between patients with low vs. high CAIX staining, as well as patients with available CAIX data vs. not. Median PFS for patients with high CAIX vs. low CAIX expression was 5.5 and 5.4 months, respectively, on the sorafenib arm (P = 0.97), and 1.5 and 1.7 months on the placebo arm (P = 0.76). Median TS for patients with high CAIX status was −14.9% vs. −12.6% in patients with low CAIX status (P = 0.63) on the sorafenib arm, and +1.3% (high CAIX) vs. +4.8% (low CAIX) in patients on the placebo arm (P = 0.60). ConclusionsDespite suggestive retrospective evidence, data from the TARGET study did not find CAIX expression status to be either predictive of clinical benefit for treatment with sorafenib or of prognostic value in patients with metastatic ccRCC following cytokine therapy.

Investigation of possible endogenous hypoxia markers in colorectal cancer

Objective: We evaluated the potential of some recently proposed hypoxia markers, being monocarboxylic acid transporter 1 (MCT1), MCT4 and prolyl hydroxylase 2 (PHD2); and a more established hypoxia marker, glucose transporter-1 (GLUT-1), by testing the association with the exogenous marker pimonidazole.Materials and methods: Paraffin embedded tumour sections of 20 colorectal cancer patients were stained for blood vessels together with either pimonidazole or carbonic anhydrase-IX (CA-IX) and single stained for MCT1, MCT4, GLUT-1, and PHD2. Expression of all markers was compared with expression of pimonidazole and micro-vessel density (MVD) and with disease-free survival (DFS) and overall survival (OS).Results: No correlation was found between the different intrinsic hypoxia markers tested and pimonidazole. A trend for high MCT1 expression in biopsies with low CA-IX expression was found (R = −0.45, p = 0.06) and also the expression of MCT1 was higher in tumours with a high MVD (R = 0.49, p = 0.04). The more advanced tumours showed a higher expression of GLUT-1 (p = 0.03). A low CA-IX expression in the tumour correlated with better DFS (p = 0.03) and related to better OS (p = 0.07).Conclusion: Although none of the tested intrinsic hypoxia markers correlated with pimonidazole staining, we confirmed the important role of both GLUT-1 and CA-IX for a more advanced pTNM (pathological tumour-node-metastasis) stage and DFS respectively.

High stromal carbonic anhydrase IX expression is associated with nodal metastasis and decreased survival in patients with surgically-treated oral cavity squamous cell carcinoma

Every year, approximately 25,000 patients are diagnosed with oral cavity squamous cell carcinoma (OCSCC) in the USA. The 5-year survival rate for OCSCC is approximately 40%. Intratumoral hypoxia confers poor prognosis and treatment failure but direct tumor oxygen measurement is challenging. Carbonic anhydrase IX (CAIX) is a marker of tissue hypoxia and we have recently shown that stromal CAIX is associated with reduced survival in patients with HPV-negative head and neck cancer. We examined the importance of this observation in OCSCC patients. We identified patients diagnosed and treated with OCSCC in Calgary (Alberta, Canada) between 1998 and 2005. Clinical and pathologic data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of archived tumor tissue. Stromal CAIX expression was assessed by quantitative immunohistochemistry (AQUA-HistoRx). The primary endpoint was disease-specific survival. We identified 102 patients with OCSCC; 87 patients had surgery as their primary treatment and adequate tumor tissue for TMA construction was available for all patients. CAIX expression was evaluable for 61 patients. High (top quartile) stromal CAIX expression was associated with significantly reduced 5-year disease-specific survival compared to low stromal CAIX expression (p<0.006). This study confirms our previously reported association between high stromal CAIX expression and significantly reduced overall survival in an independent, predominantly p16-negative, cohort of surgically treated OCSCC. Assessment of stromal CAIX expression could identify patients with the least favorable prognosis and inform therapeutic strategies in OCSCC.

Carbonic anhydrase IX and pathological features as predictors of outcome in patients with metastatic clear‐cell renal cell carcinoma receiving vascular endothelial growth factor‐targeted therapy

To investigate the utility of tumour carbonic anhydrase IX (CAIX) expression and histological features for predicting the outcome in patients with metastatic clear-cell renal cell carcinoma (mRCC) treated with vascular endothelial growth factor (VEGF)-targeted therapy. We identified 118 patients with mRCC initiating first-line VEGF-targeted therapy, including 94 with clinical and histological data, and available tissue. The primary endpoint was to detect an interaction between sorafenib vs sunitinib treatment and CAIX status on tumour shrinkage. Other treatment outcomes were also assessed. There was heterogeneity in tumour responsiveness to sunitinib or sorafenib according to CAIX status; the mean shrinkage was -17% vs -25% for sunitinib-treated patients with high vs low tumour CAIX expression, compared to -13% vs +9% for sorafenib-treated patients (P interaction, 0.05). A higher tumour clear-cell component was independently associated with greater tumour shrinkage (P= 0.02), response (P= 0.02) and treatment duration (P= 0.02). Although CAIX expression had no prognostic value in patients with clear-cell mRCC treated with VEGF-targeted therapy, it might be a predictive biomarker for response to sorafenib treatment. Patients with a higher clear-cell component in their tumours are likely to have a superior clinical benefit from VEGF-targeted therapy.

Co‐expression of Hif1α and CAIX is associated with poor prognosis in oral squamous cell carcinoma patients

This study investigates the prognostic impact of the expression of hypoxia-inducible factor 1alpha (Hif1alpha) and carbonic anhydrase IX (CAIX) detected by immunohistochemistry in oral squamous cell carcinoma (OSCC). Statistical analysis of immunohistochemical results with clinical parameters including survival outcomes was performed for 80 OSCC patients. Patients with a low expression of both proteins survived on average 54.8 months, whereas those with an increased expression of Hif1alpha in their tumors combined with a low expression of CAIX survived on average only 37.6 months (P = 0.026). In multivariate Cox's regression hazard analysis, again patients with a low expression of Hif1alpha/CAIX had the best prognosis, whereas patients with increased Hif1alpha and low CAIX expression carried a 4.97-fold increased risk of tumor-related death (P = 0.042). A co-detection of low Hif1alpha/CAIX expression is significantly correlated with a better prognosis for OSCC patients, which may have implications for therapy options for these patients.

Prognostic relevance of carbonic anhydrase-IX in high-risk, early-stage cervical cancer: A Gynecologic Oncology Group study

Objectives This study aimed to determine whether carbonic anhydrase-IX (CA-IX) was associated with progression-free survival (PFS) and overall survival (OS) in women with high-risk, early-stage cervical cancer treated with adjuvant pelvic radiotherapy with or without radiosensitizing chemotherapy. Methods CA-IX expression was detected using an immunohistochemistry assay and categorized as low when ≤ 80% of tumor cells exhibited CA-IX staining and high when > 80% tumor cells display CA-IX staining. Associations between CA-IX expression and clinical characteristics, angiogenesis marker expression, and clinical outcome were evaluated. Results High CA-IX expression was observed in 35/166 (21.1%) of cases. CA-IX expression was not associated with age, race, stage, cell type, grade, positive margins, parametrial extensions, positive lymph nodes, or lymphovascular space invasion but was associated with tumor size categorized as < 2 , 2–2.9 , or ≥ 3 cm (high expression: 4.7% vs. 23.2% vs. 32.5%, P = 0.003) and cervical invasion confined to the inner two-thirds compared with the outer third of the cervix (high expression: 6.1% vs. 23.7%, P = 0.028). CA-IX expression was not associated with immunohistochemical expression of p53, CD31, CD105, thrombospondin-1, or vascular endothelial growth factor-A. Women with high versus low CA-IX expression had similar PFS ( P = 0.053) and significantly worse OS ( P = 0.044). After adjusting for prognostic clinical covariates, high CA-IX expression was an independent prognostic factor for PFS (hazard ratio [HR] = 2.12; 95% confidence interval [CI] = 1.13–3.95; P = 0.019) and OS (HR = 2.41; 95% CI = 1.24–4.68; P = 0.009). Conclusions Tumor hypoxia measured by immunohistochemical expression of CA-IX is an independent prognostic factor for both PFS and OS in high-risk, early-stage cervical cancer.

Prognostic and predictive values of carbonic anhydrase IX (CAIX) and pathologic features in patients with metastatic clear cell renal cell carcinoma receiving targeted therapy

e16067 Background: Tumor Carbonic Anhydrase IX (CAIX) expression and histologic features can predict outcome in patients with metastatic renal cell carcinoma (mRCC) treated with immunotherapy. We sought to investigate the prognostic and predictive utility of such features in patients receiving VEGF-targeted therapy. Methods: We identified 118 patients with mRCC initiating first- line VEGF-targeted therapy including 94 with clinical data, clear cell histology and available tissue. Tumors were evaluated for specific histologic features and for CAIX expression by immunohistochemistry using the MN75 antibody. The relationship between these pathology findings and tumor shrinkage and other treatment outcomes was assessed. Results: Higher tumor clear cell component was independently associated with greater tumor shrinkage (p=0.02), response (p=0.02) and treatment duration (p=0.02). Patients with high vs. low tumor CAIX expression had mean tumor shrinkages of -12% vs. -5%, respectively (p=.38). There was heterogeneity in tumor responsiveness to sunitinib or sorafenib according to CAIX status (p=0.055 for interaction): mean shrinkage was -17% vs. -25% (mean difference +8%, 95% CI -14% to +31%) for sunitinib-treated patients with high vs. low tumor CAIX expression compared to -13% vs. +9% (mean difference -22%, 95% CI -42% to -1%) for sorafenib-treated patients. Conclusions: Patients with higher clear cell component in their tumors are likely to experience superior clinical benefit from VEGF-targeted therapy. Although CAIX expression was not found to be of prognostic value in patients with clear cell mRCC treated with VEGF-targeted therapy, it may be a predictive biomarker for response to sorafenib treatment. [Table: see text]

Carbonic Anhydrase IX Expression in Clear Cell Renal Cell Carcinoma

Carbonic anhydrase IX (CAIX)-a protein maintaining intracellular and extracellular pH-reportedly also influences regulation of cell proliferation, oncogenesis, and tumor progression. Its expression is von Hippel-Lindau-hypoxia inducible factor pathway dependent. Immunohistochemical (IHC) studies show that CAIX is diffusely overexpressed in clear cell renal cell carcinoma (CRCC), making it a potentially important differential diagnostic marker. Prognostically, low CAIX expression reportedly indicates poor survival and low response to interleukin therapy in CRCC. Most IHC studies have used clone M75 as the primary antibody, which is not commercially available. We evaluated a new commercially available antibody (clone NB100-417) to assess its expression in CRCC and compared its results with M75. On a tissue microarray of CRCC, IHC staining was performed using both antibodies. The immunoreactivity was graded as 0; 1+, 1% to 25%; 2+, 26% to 50%; and 3+, >50% tumor cells immunoreactive. Ninety-one out of ninety-five (96%) cases showed similar staining grade with excellent agreement (kappa=0.65, weighted kappa=0.75). Grade 3+ expression of CAIX was observed in 89 (94%) cases each, with both antibodies. Both antibodies produced intense membrane-predominant expression, limited to tumor cells. More than 95% of the tumors with low nuclear grade, compared with 84% and 88% of tumors with high nuclear grade, showed 3+ expression using both antibodies. Thus, most CRCC show strong and diffuse expression of CAIX, and the expression is comparable using both antibodies. Therefore, similar to the clone M75, NB100-417 can be used as a diagnostic and potentially a prognostic marker in CRCC, with the advantage of its commercial availability.

Carbonic Anhydrase IX Is Not an Independent Predictor of Outcome for Patients With Clear Cell Renal Cell Carcinoma

Expression of carbonic anhydrase IX (CAIX) has been reported to be an independent predictor of outcome and is being investigated as a therapeutic target for patients with clear cell renal cell carcinoma (ccRCC). We attempted to validate the prognostic utility of CAIX expression using a large cohort of ccRCC patients with long-term follow-up. We identified 730 patients with unilateral, sporadic ccRCC treated surgically between 1990 and 1999. Anti-CAIX monoclonal antibody (clone M75) was used, and tumor specimens were blindly scored for expression levels. Associations of CAIX expression with RCC death were evaluated using Cox proportional hazards regression models. There were 241 RCC deaths and a median of 9.4 years of follow-up for patients still under observation. CAIX was expressed in 708 (97.0%) of the specimens; 163 tumors (22.3%) exhibited low ( 85% tumor cells positive) expression, and 567 (77.7%) exhibited high (> 85% tumor cells positive) expression. Univariately, low CAIX expression was associated with increased risk of RCC death relative to high expression (risk ratio = 1.65; P < .001). However, low CAIX expression was not associated with RCC death after adjusting for nuclear grade or coagulative tumor necrosis. Additionally, we observed CAIX expression in a number of extrarenal organs. CAIX is strongly expressed by ccRCC. Although CAIX is associated with outcome in patients with ccRCC, it is not an independent prognostic marker. Furthermore, CAIX expression is apparent in extrarenal organs. As such, exploitation of CAIX as a prognostic marker and therapeutic target merits additional consideration.

Prospective evaluation of carbonic anhydrase IX (CAIX) as a molecular marker in metastatic renal cell carcinoma

5112 Background: CAIX is an important molecular marker of survival and response to immunotherapy in patients with metastatic renal cell carcinoma (RCC). The purpose of this study was to prospectively evaluate the performance of CAIX in patients with metastatic RCC. Methods: This study accrued 32 consecutive patients who were treated for metastatic clear cell RCC between January 2004 and May 2006. Immunohistochemical staining of the primary tumor was performed using the mouse monoclonal antibody MN-75. Patients were stratified into two groups: high CAIX expression (&gt;85%) and low CAIX expression (=85%) according to the percentage of cells staining positive for CAIX. Study endpoints included disease-specific survival time (DSS) and response to treatment according to RECIST criteria. Results: Four (12.5%), 21 (65.6%), and 7 patients (21.9%) were classified into low risk, intermediate risk, and high risk groups according to the University of California Integrated Staging System (UISS). Twenty (62.5%) of the 32 patients had high CAIX expression. The median follow-up was 11.4 months. Patients with low CAIX expression had significantly worse prognosis than patients with high CAIX expression (median survival: 15.2 months vs. not reached, p=0.01, 1-year DSS rate: 63% vs. 83%) and a 3.9 fold increased risk of death from RCC (95% CI, 1.2–12.7). All 4 patients in the low risk UISS group had high CAIX expression, and all were alive at 1 year. Nine patients received high dose IL-2 based immunotherapy, including 8 who had high CAIX expression, and a 1-year DSS rate of 87.5%. Of the patients expressing high CAIX, 3 (37.5%) were responders to IL-2 including 1 partial and 2 complete responses, 3 (37.5%) exhibited stable disease and 2 (25.0%) progressed during treatment. Conclusions: The results of this first prospective study of CAIX in metastatic RCC confirm that high CAIX expression is associated with better survival and enhanced response to IL-2 based immunotherapy. Patients with high CAIX expression, specifically those with low risk RCC, should be considered candidates to receive immunotherapy, whereas patients with low CAIX expression or in higher risk groups may be better candidates for targeted or other experimental therapy. No significant financial relationships to disclose.