Placental SARS-CoV-2 distribution correlates with level of tissue oxygenation in COVID-19-associated necrotizing histiocytic intervillositis/perivillous fibrin deposition.

INTRODUCTION: Recent evidence supports the - rare - occurrence of vertical transplacental SARS-CoV-2 transmission. We previously determined that placental expression of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, and associated viral cell entry regulators is upregulated by hypoxia. In the present study, we utilized a clinically relevant model of SARS-CoV-2-associated chronic histiocytic intervillositis/massive perivillous fibrin deposition (CHIV/MPFVD) to test the hypothesis that placental hypoxia may facilitate placental SARS-CoV-2 infection. METHODS: We performed a comparative immunohistochemical and/or RNAscope in-situ hybridization analysis of carbonic anhydrase IX (CAIX, hypoxia marker), ACE2 and SARS-CoV-2 expression in free-floating versus fibrin-encased chorionic villi in a 20-weeks' gestation placenta with SARS-CoV-2-associated CHIV/MPVFD. RESULTS: The levels of CAIX and ACE2 immunoreactivity were significantly higher in trophoblastic cells of fibrin-encased villi than in those of free-floating villi, consistent with hypoxia-induced ACE2 upregulation. SARS-CoV-2 showed a similar preferential localization to trophoblastic cells of fibrin-encased villi. DISCUSSION: The localization of SARS-CoV-2 to hypoxic, fibrin-encased villi in this placenta with CHIV/MPVFD suggests placental infection and, therefore, transplacental SARS-CoV-2 transmission may be promoted by hypoxic conditions, mediated by ACE2 and similar hypoxia-sensitive viral cell entry mechanisms. Understanding of a causative link between placental hypoxia and SARS-CoV-2 transmittability may potentially lead to the development of alternative strategies for prevention of intrauterine COVID-19 transmission.

Synthesis and evaluation of novel technetium-99m-hydroxamamide complex based on imidazothiadiazole sulfonamide targeting carbonic anhydrase-IX for tumor imaging.

Carbonic anhydrase-IX (CA-IX) is an attractive target for detecting tumors associated with a poor prognosis. We previously reported a [99mTc]hydroxamamide complex based on ureidosulfonamide as a CA-IX ligand ([99mTc]URB2A), which showed a favorable affinity for CA-IX high-expressing cells in vitro and tumors in vivo; however, radioactivity retention in the blood pool suggested a high background signal on imaging. To improve the pharmacokinetics of [99mTc]URB2A, in this study, we designed and synthesized [99mTc]ISB2 based on imidazothiadiazole sulfonamide, which exhibited greater CA-IX affinity and faster clearance from the blood pool than ureidosulfonamide in studies using corresponding 111In-labeled compounds, and evaluated its utility for CA-IX imaging. In an in vitro cell binding assay, [99mTc]ISB2 markedly bound to CA-IX high-expressing (HT-29) cells; moreover, its binding was greater than that of [99mTc]URB2A. In an in vivo biodistribution assay, [99mTc]ISB2 showed faster clearance from the blood pool than [99mTc]URB2A; however, lower HT-29 tumor accumulation was observed. Further structural modification of [99mTc]ISB2 to improve its stability may lead to the development of a useful [99mTc]hydroxamamide complex for CA-IX imaging.

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Synthesis and evaluation of indium-111-labeled imidazothiadiazole sulfonamide derivative for single photon emission computed tomography imaging targeting carbonic anhydrase-IX.

Carbonic anhydrase-IX (CA-IX) is a zinc enzyme overexpressed in the hypoxic regions of many types of solid tumors; therefore, in vivo imaging of CA-IX may contribute to cancer diagnosis. In this study, we newly designed and synthesized an 111In-labeled CA-IX imaging agent based on an imidazothiadiazole sulfonamide (IS) scaffold conjugated with a chelating moiety, DO3A ([111In]DO3A-IS1), and evaluated its utility for imaging of CA-IX high-expressing tumors. [111In]DO3A-IS1 was successfully synthesized at a 76% radiochemical yield by reacting its precursor with 111InCl3 in acetate buffer. In in vitro assays, [111In]DO3A-IS1 showed marked stability in murine plasma and greater binding to CA-IX high-expressing (HT-29) cells (118 ± 21% initial dose/mg protein) than CA-IX low-expressing (MDA-MB-231) cells (1.4 ± 0.3% initial dose/mg protein). Moreover, in an in vivo biodistribution assay, [111In]DO3A-IS1 showed marked accumulation in the HT-29 tumor (8.71 ± 1.41% injected dose/g at 24 h postinjection). In addition, in a single photon emission computed tomography (SPECT) study, [111In]DO3A-IS1 clearly and selectively visualized the HT-29 tumor as compared with the MDA-MB-231 tumor. These results indicate that [111In]DO3A-IS1 may serve as a useful SPECT imaging agent with the novel scaffold targeting CA-IX.

ROC-1, P21 and CAIX as markers of tumor aggressiveness in bladder carcinoma in Egyptian patients.

BACKGROUND: Bladder cancer (BC) is one of the most common malignancies in Egypt, representing about 8.7% of cancers in both sexes with more predominance in males, making identification of valuable predictive and prognostic markers, mandatory. Cullin-RING ligases (CRL) play an important role in the ubiquitination of cell cycle-related proteins or other proteins (e.g., DNA replication protein, signal transduction protein). Regulator of Cullins-1 (ROC-1) is a key subunit of CRL. P21 belongs to the family of cyclin dependent kinase inhibitors (CKIs) which regulates cell cycle by inactivating Cyclin- Dependent Kinases key regulators of the cell cycle. CAIX a highly active member of the family of carbonic anhydrases has gained much interest as a hypoxic marker. Hypoxia is a consequence of the rapid growth of many tumors, including bladder cancer, and is an important regulator of gene expression and resistance to chemotherapy and radiotherapy. Therefore the purpose of this study is to evaluate the role of ROC-1, CAIX and P21 and its relationship with the clinico-pathological features of bladder cancer in Egyptian patients. METHODS: Using the standard immunohistochemical technique, ROC-1, CAIX and P21 expression in 80 primary bladder carcinomas and 15 normal bladder specimens as control group were assessed. The bladder carcinoma cases included 50 cases with muscle invasive bladder cancer and 30 cases with non-muscle invasive bladder cancer. RESULTS: Over expression of ROC-1, CAIX and P21 in BC were significantly associated with muscularis propria invasion and high grade BC. ROC-1, CAIX and P21, showed significant inverse relationship in primary BC cases. CAIX expression was significantly higher in BC compared with controls. Regarding the survival analysis, expression of ROC-1, CAIX and P21 didn't affect the survival of BC patients. CONCLUSIONS: High expression of ROC-1, CAIX and P21 could be promising potential biomarkers for identifying patients with poor prognostic factors in bladder cancer serving as potential targets for cancer therapy.

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

BACKGROUND: Low 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. METHODS: A 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). RESULTS: There 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. CONCLUSION: The 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.

Carbonic anhydrase 9 immunohistochemistry as a tool to predict or validate germline and somatic VHL mutations in pheochromocytoma and paraganglioma-a retrospective and prospective study.

The development of pheochromocytomas and paragangliomas is strongly linked to the presence of germline mutations in more than 15 predisposing genes. Among them, germline and somatic VHL mutations account for ~10% of all cases. In contrast with SDHA and SDHB immunohistochemistries that are routinely used to validate SDHx gene mutations, there is no such tool available for VHL mutations. The aim of this study was to evaluate whether CA9 immunostaining could be used as a tool to predict the presence or validate the pathogenicity of VHL gene mutations in paraganglioma. Immunohistochemistry for CA9 was performed on 207 tumors. A retrospective series of 100 paragangliomas with known mutation status for paraganglioma susceptibility genes was first investigated. Then, a prospective series of 107 paragangliomas was investigated for CA9 immunostaining followed by germline and/or somatic genetic testing of all paraganglioma susceptibility genes by next-generation sequencing. Cytosolic CA9 protein expression was heterogeneous in the different samples. However, we observed that a membranous CA9 staining was almost exclusively observed in VHL-related cases. Forty two of 48 (88%) VHL-mutated samples showed a CA9 membranous immunostaining. Positive cells were either isolated, varying from 1 or 2 cells (5% of cases) to 10-20 cells per tumor block (35% of cases), grouped in areas of focal positivity representing between 1 and 20% of the tissue section (35% of cases), or widely distributed on 80-100% of the tumor sections (25% of samples). In contrast, 142/159 (91%) of non-VHL-mutated tumors presented no membrane CA9 localization. Our results demonstrate that VHL gene mutations can be predicted or validated reliably by an easy-to-perform and low-cost immunohistochemical procedure. CA9 immunohistochemistry on paragangliomas will improve the diagnosis of VHL-related disease, which is important for the surveillance and therapeutic management of paraganglioma patients, and in case of germline mutation, their family members.

Poor outcome in hypoxic endometrial carcinoma is related to vascular density.

BACKGROUND: Identification of endometrial carcinoma (EC) patients at high risk of recurrence is lacking. In this study, the prognostic role of hypoxia and angiogenesis was investigated in EC patients. METHODS: Tumour slides from EC patients were stained by immunofluorescence for carbonic anhydrase IX (CAIX) as hypoxic marker and CD34 for assessment of microvessel density (MVD). CAIX expression was determined in epithelial tumour cells, with a cut-off of 1%. MVD was assessed according to the Weidner method. Correlations with disease-specific survival (DSS), disease-free survival (DFS) and distant disease-free survival (DDFS) were calculated using Kaplan-Meier curves and Cox regression analysis. RESULTS: Sixty-three (16.4%) of 385 ECs showed positive CAIX expression with high vascular density. These ECs had a reduced DSS compared to tumours with either hypoxia or high vascular density (log-rank p = 0.002). Multivariable analysis showed that hypoxic tumours with high vascular density had a reduced DSS (hazard ratio [HR] 3.71, p = 0.002), DDFS (HR 2.68, p = 0.009) and a trend for reduced DFS (HR 1.87, p = 0.054). CONCLUSIONS: This study has shown that adverse outcome in hypoxic ECs is seen in the presence of high vascular density, suggesting an important role of angiogenesis in the metastatic process of hypoxic EC. Differential adjuvant treatment might be indicated for these patients.

Fluorine-18 click radiosynthesis and microPET/CT evaluation of a small peptide-a potential PET probe for carbonic anhydrase IX.

Carbonic anhydrase IX (CA IX) is the first carbonic anhydrase found to be associated with cancer that is over-expressed in a variety of human solid tumors. As a surrogate marker for hypoxia, the expression of CA IX is strongly upregulated in hypoxic tumors by hypoxia and hypoxia-inducible factor 1a (HIF-1a). In our pursuit of a CA IX-specific PET probe, we designed and synthesized a peptide-based CA IX imaging probe by the efficient click reaction of 1,3-dipolar cycloaddition of terminal alkynes and organic azides. The probe 18F-CA IX-P1-4-10 was obtained with a radiochemical yield of 35-45% (n = 5) and radiochemical purity of >99% in 70-80 min (HPLC purification time included). 18F-CA IX-P1-4-10 had good stability in phosphate buffered saline (PBS), but about 51% peptide degradation was detected in new-born calf serum (NBCS) after incubation. Preliminary microPET/CT experiments demonstrated a specific uptake of 18F-CA IX-P1-4-10 in HT29 tumor and the uptake of 18F-CA IX-P1-4-10 was blocked by peptide CA IX-P1-4-10-Yne pretreatment. Immunohistochemical staining and western blotting studies confirmed the HT29 tumor was CA IX-positive which further proved tumor accumulation of 18F-CA IX-P1-4-10 was correlated with CA IX expression. The results suggest that 18F-CA IX-P1-4-10 is a promising PET tracer for the specific imaging of CA IX-expressing tumors at the molecular level.

CAIX and pax-8 Commonly Immunoreactive in Endolymphatic Sac Tumors: A Clinicopathologic Study of 26 Cases with Differential Considerations for Metastatic Renal Cell Carcinoma in von Hippel-Lindau Patients.

Endolymphatic sac tumors (ELSTs) are rare, slowly growing temporal bone neoplasms which show a high association with von Hippel-Lindau (VHL) syndrome. The immunohistochemistry evaluation of these papillary-cystic neoplasms frequently raises the differential diagnosis with renal cell carcinoma, among other metastatic neoplasms, whether in VHL patients or not. A cohort of 26 patients with ELSTs were evaluated for histologic features, immunohistochemistry findings, and association with VHL. Standard immunohistochemistry evaluation was performed. Sixteen females and 10 males ranging in age from 10 to 69 years (mean 44; VHL mean: 32) at initial presentation, comprised the cohort of patients. Most (86%) experienced hearing changes or inner ear symptoms (vertigo, dizziness), with an average duration of symptoms for 39 months (range 2-240 months). The tumors were an average of 2.9 cm (range 0.4-8 cm), with 14 left, 11 right sided and one bilateral tumor. Nine patients had documented VHL, with 3 patients having a concurrent or subsequent clear cell renal cell carcinoma. Patients were followed an average of 6.2 years (available in 24 patients): 19 alive without disease, 7.5 years; 2 dead without disease, 1.2 years; and 3 alive with disease, 3.1 years. The neoplastic cells show the following immunohistochemistry findings: AE1/AE3, EMA, CK7, CAIX, GLUT1, VEGF: 100% of cases tested were positive; pax-8: 85% of cases positive; CD10 and RCC: 0% of cases reactive. Based on this cohort of 26 patients with ELST, 9 of whom had VHL, the strong pax-8 and CAIX should be used in conjunction with negative CD10 and RCC to help exclude a metastatic renal cell carcinoma. As CAIX is an enzyme overexpressed in hypoxia and hypoxia inducible factor is what VHL protein regulates, this is an expected, although previously unreported finding. Whether part of VHL or not, VHL mutations may be a somatic rather than germline finding in the tumors, a possible further explanation for the CAIX reaction.

Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype.

Bladder cancer has the highest recurrence rate of all cancers due in part to inadequate transurethral resection. Inadequate resection is caused by the inability of cystoscopes to detect invisible lesions during the resection procedure. To improve detection and resection of nonmuscle invasive bladder cancer, we quantified the ability of a surface-enhanced Raman nanoparticle and endoscope system to classify bladder tissue as normal or cancerous. Both antibody-based (active) and tissue permeability-based (passive) targeting mechanisms were evaluated by topically applying nanoparticles to ex vivo human bladder tissue samples. Multiplexed molecular imaging of CD47 and Carbonic Anhydrase 9 tumor proteins gave a receiver operating characteristic area under the curve (ROC AUC of 0.93 (0.75, 1.00). Furthermore, passively targeted nanoparticles enabled tissue classification with an ROC AUC of 0.93 (0.73, 1.00). Passively targeted nanoparticles penetrated 5-fold deeper and bound to tumor tissue at 3.3-fold higher concentrations in cancer compared to normal bladder urothelium, suggesting the existence of an enhanced surface permeability and retention effect in human bladder cancer.

Prognostic significance of carbonic anhydrase IX overexpression in stage III non-small cell lung cancer patients after neoadjuvant treatment.

BACKGROUND: To assess the prognostic importance of carbonic anhydrase IX (CA IX), a hypoxic biomarker, after neoadjuvant treatment in Stage III non-small cell lung cancer (NSCLC) patients. METHODS: Tissue CA IX expression was examined after surgical resection in 77 patients who had undergone neoadjuvant treatment. The effects of CA IX overexpression and other clinical factors on disease-free survival and overall survival were investigated. RESULTS: In multivariate analysis, number of neoadjuvant chemotherapy (CT) courses and gender emerged as significant independent predictors for disease-free survival, where administration of 2-3 courses of neoadjuvant chemotherapy (CT) (HR, 3.2 [95% CI 1.3-7.6], p = 0.009) and female gender were associated with poor survival (HR, 3.2 [95% CI 1.3-7.7], p = 0.009). The only significant independent predictor for overall survival was recurrence (HR, 5.6 [95% CI 2.4-12.8], p < 0.001). On the other hand, CA IX overexpression was not associated with disease free survival (p = 0.560) or overall survival (p = 0.799). DISCUSSION: Our results do not suggest a prognostic role for CA IX overexpression in stage III NSCLC patients who received neoadjuvant treatment.

Carbonic anhydrase IX is a prognostic biomarker in glioblastoma multiforme.

The identification of prognostic factors in patients with glioblastoma multiforme (GBM) represents an area of increasing interest. Carbonic anhydrase IX (CA-IX), a hypoxia marker, correlates with tumor progression in a variety of human cancers. However, the role of CA-IX in GBM remains largely unknown. In the present study, we evaluated the prognostic role of CA-IX in GBM patients. In total, 66 consecutive patients with GBM who received concomitant chemoradiotherapy and adjuvant chemotherapy with temozolomide were retrospectively reviewed, and all patients received temozolomide chemotherapy for at least 3 months. Kaplan-Meier curves and log-rank tests were used for analysis of progression-free survival (PFS) and overall survival (OS), and a multivariate Cox proportional hazard model was employed to identify factors with an independent effect on survival. The median OS was longer in patients with low levels of CA-IX expression (18 months) compared to patients overexpressing CA-IX (9 months) (P = 0.004). There was not a statistically significant difference in median PFS (3.5 vs. 8 months, P = 0.054) between patients with high or low levels of CA-IX expression. In multivariate analysis, the variables that were identified as significant prognostic factors for OS were preoperative Karnofsky performance scale score (KPS) (hazard ratio (HR), 3.703; P = 0.001), CA-IX overexpression (HR, 1.967; P = 0.019), and incomplete adjuvant temozolomide treatment (HR, 2.241; P = 0.003) and gross-total resection (HR, 1.956; P = 0.034). Our findings indicated that CA-IX may be a potential prognostic biomarker in the treatment of GBM.

Carbonic anhydrase 9 expression in well-differentiated pancreatic neuroendocrine neoplasms might be associated with aggressive behavior and poor survival.

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 (< 1 cm) 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 (< 1 cm) 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.

Versatile protein recognition by the encoded display of multiple chemical elements on a constant macrocyclic scaffold.

In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody-antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

Acetazolamide-based [18F]-PET tracer: In vivo validation of carbonic anhydrase IX as a sole target for imaging of CA-IX expressing hypoxic solid tumors.

Carbonic anhydrase IX is overexpressed in many solid tumors including hypoxic tumors and is a potential target for cancer therapy and diagnosis. Reported imaging agents targeting CA-IX are successful mostly in clear cell renal carcinoma as SKRC-52 and no candidate was approved yet in clinical trials for imaging of CA-IX. To validate CA-IX as a valid target for imaging of hypoxic tumor, we designed and synthesized novel [18F]-PET tracer (1) based on acetazolamide which is one of the well-known CA-IX inhibitors and performed imaging study in CA-IX expressing hypoxic tumor model as 4T1 and HT-29 in vivo models other than SKRC-52. [18F]-acetazolamide (1) was found to be insufficient for the specific accumulation in CA-IX expressing tumor. This study might be useful to understand in vivo behavior of acetazolamide PET tracer and can contribute to the development of successful PET imaging agents targeting CA-IX in future. Additional study is needed to understand the mechanism of poor targeting of CA-IX, as if CA-IX is not reliable as a sole target for imaging of CA-IX expressing hypoxic solid tumors.

Overexpression of FZD1 and CAIX are Associated with Invasion, Metastasis, and Poor-Prognosis of the Pancreatic Ductal Adenocarcinoma.

Approximately 80% of patients with pancreatic ductal adenocarcinoma (PDAC) have metastatic disease with poor prognosis, but clinically available biomarkers have not yet been identified. This study was to investigate the clinical significance of FZD1 and CAIX in PDACs. FZD1 and CAIX protein expression was measured using EnVision immunohistochemistry. Positive FZD1 or CAIX expression was significantly higher in PDAC than that in precursor lesions (p < 0.01). Positive FZD1 or CAIX expression was significantly lower in cases with well-differentiated adenocarcinoma, no-metastasis of the lymph node, no-invasion of regional tissues, and TNM I/II stage disease than in cases with poorly-differentiated adenocarcinoma, metastasis and invasion, and TNM stage III+ IV stage disease (p < 0.05 or p < 0.01). The expression of FZD1 positively correlated with CAIX in PDAC (P = 0.000). Univariate Kaplan-Meier analysis showed that FZD1 and/or CAIX expression (p < 0.001) was significantly associated with shorter overall survival (p < 0.05). Cox multivariate analysis showed that differentiation, tumor mass, lymph node metastasis, invasion, TNM stage, FZD1 and CAIX levels negatively correlated with overall survival. Positive FZD1 and CAIX expressions are poor prognostic factors in PDAC patients. FZD1 and CAIX might be important biological markers for the carcinogenesis, metastasis, invasion, and prognosis of PDAC.

Selecting patients for hyperthermia combined with preoperative chemoradiotherapy for locally advanced rectal cancer.

BACKGROUND: This study investigated the role of hyperthermia combined with preoperative concurrent chemoradiotherapy (CCRT) for locally advanced rectal cancer (LARC) according to hypoxic marker expression. METHODS: One hundred and nine LARC patients with tissue blocks available for immunohistochemical assessment of carbonic anhydrase 9 (CA9) expression were reviewed. CA9 expression was considered positive when the staining percentage of tumor cells was >25% (n = 31). Pelvic radiotherapy with a total dose of 39.6-45 Gy was delivered concurrently with fluorouracil-based chemotherapy. Hyperthermia was administered to 52 patients twice a week during CCRT. Treatment response and outcomes were compared between hyperthermochemoradiotherapy (HCRT) and CCRT groups. RESULTS: In patients with positive CA9 expression, the rates of downstaging (p = 0.060) and pathologic complete response (p = 0.064) tended to be higher in the HCRT group than in the CCRT group. Distant metastasis-free survival (p = 0.029) and cancer-specific survival (p = 0.020) were significantly worse in tumors with both positive CA9 expression and poor tumor response. Negative CA9 expression, presence of major tumor response, and the use of hyperthermia were significant favorable prognostic factors for cancer-specific survival after the first recurrence in multivariate analysis. CONCLUSIONS: Hyperthermia might selectively enhance the preoperative treatment response in LARC with positive CA9 expression and offset the negative effect of hypoxia on prognosis. Pretreatment evaluation of hypoxia could aid in the selection of patients who might benefit from hyperthermia.

Identification and validation of novel prognostic markers in Renal Cell Carcinoma.

Kidney cancer (Renal Cell Carcinoma (RCC)) is one of the most deadly malignancies due to frequent late diagnosis and poor treatment options. Histologically, RCC embraces a wide variety of different subtypes with the clear cell variant (ccRCC) being the most common, accounting for 75-90% of all RCCs. At present, the surveillance protocols for follow-up of RCC patients after radical nephrectomy are based on the American Joint Committee on Cancers (AJCC) pathological tumor-node-metastasis (TNM) classification system. Other comprehensive staging modalities have emerged and have been implemented in an attempt to improve prognostication by combining other pathological and clinical variables, including Fuhrman nuclear grade and Leibovich score. However, even early stage tumors remain at risk of metastatic progression after surgical resection and 20-40% of patients undergoing nephrectomy for clinically localized RCC will develop a recurrence. Identifying this high-risk group of RCC patients remains a challenge. Hence, novel molecular prognostic biomarkers are needed to better predict clinical outcomes. An intensive search within this field has been ongoing in the past few years, and the three main predictive and prognostic markers validated in RCC are Von Hippel Lindau (VHL), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CAIX). Nonetheless, the use of these is still debated and none of them have yet been implemented in clinical routine. RCC is resistant to conventional oncological therapies, such as chemotherapy and radiation. The availability of novel targeted therapies directed against tumorigenic and angiogenic pathways have increased over the last years, and the outcome of patients with advanced RCC has significantly improved as a consequence. Unfortunately, all patients eventually become resistant. Thus, the development of novel targeted therapies is of great importance. The aim of this thesis was therefore to contribute in the search for novel prognostic molecular markers in RCC and to identify novel targeted therapies by in-vitro studies. This was specifically conducted by investigating; 1) The impact of symptom presentation of RCC on prognosis, 2) The expression of Calcium-activated potassium channels in RCC, the correlation of KCa3.1 to prognosis in ccRCC and the ability of TRAM-34, RA-2 and Paxilline to inhibit the proliferation of ccRCC cell lines in-vitro, 3) The gene expression and prognostic value of 19 selected genes in ccRCC and 4) The expression of the protein kinase CK subunits in subtypes of RCC, the prognostic impact of high protein expression of the CK2α subunit in ccRCC and the ability of CX-4945 and E9 to inhibit ccRCC growth in-vitro. Our molecular study cohort consisted of 155 patients with different subtypes of RCC and the benign renal neoplasm, oncocytoma. They were diagnosed in Region of Southern Denmark in 2001-2013. Frozen tissue from tumor and normal renal cortex parenchyma, together with paraffin-embedded tissue was available for every patient. We performed gene expression analysis by qRT-PCR, immunohistochemical staining of Tissue Micro Arrays, protein kinase activity analysis and functional studies. Study I was performed as a descriptive observational study focusing on the prognostic impact of symptom presentation in RCC. We included 204 patients with renal neoplasms diagnosed in 2011-2012. Incidentally discovered RCC without symptomatic presentation had overall a better prognosis, and presented with smaller tumors, a lower T-stage, lower Fuhrman grade and lower Leibovich score. In addition, the non-symptomatic patient group experienced metastatic disease less frequently. In study II we focused on the expression of two calcium-activated potassium channels in ccRCC and oncocytoma. Both KCa3.1 and KCa1.1 were higher expressed in ccRCC compared to oncocytoma. High expression of KCa3.1 was moreover correlated with poor progression free survival of ccRCC. Functional studies provided new insights since we could detect currents compatible with KCa3.1 and KCa1.1 in the cell membrane of primary and commercial ccRCC cell lines. Nonetheless, we were not able to show any significant inhibition of cell growth by the selective inhibitors of KCa3.1 and KCa1.1, TRAM-34, RA-2 and Paxilline. In study III our aim was to investigate the prognostic role of 19 genes selected on the basis of an earlier study done by the group. We used Taqman® Low Density Array to perform a quantitative real-time PCR analysis. By selecting an optimal cut-point and correct for overestimation of the p-value, we could identify three genes with impact on prognosis of ccRCC in both univariate and multivariate analysis. High expression of the genes SPP1 and CSNK2A1 (encoding Osteopontin and CK2α respectively) correlated with poor prognosis while high expression of DEFB1 (encoding ß-Defensin) correlated with better prognosis. Study IV focused on validating the results obtained in Paper III by investigating the protein expression of CK2α (Protein kinase 2, alpha subunit) in the different subtypes of RCC and oncocytoma. Furthermore, we investigated whether protein expression of CK2α in ccRCC correlated with prognosis. Here we could show, that a positive nuclear staining was a marker of poor prognosis in high-stage ccRCC. Moreover, enzyme activity analysis revealed a higher activity of the protein kinase in tumor tissue of ccRCC than in normal renal cortex. Novel insights were provided in a proliferation study where we investigated the selective inhibitors of CK2α, CX-4945 and E9. CX-4945 was able to inhibit ccRCC cell growth by nearly 50%. All together the studies presented in this thesis add additional information to the ongoing research within identification of novel prognostic markers in ccRCC. We have discovered four new molecular markers, which reliably can predict prognosis at the time of diagnosis. Additionally, we identified CK2α as a novel therapeutic target of ccRCC. The studies suggest further research to validate the findings on larger cohorts and thereby obtain more insight into the involved pathways. Future research initiatives based on the results presented in this thesis could clarify the potential role of CX-4945 as a novel targeted treatment of ccRCC patients.