Sounak Gupta

Magnetic Resonance Elastography. A Novel Technique for the Detection of Hepatic Fibrosis and Hepatocellular Carcinoma After the Fontan Operation

OBJECTIVE To evaluate the utility of magnetic resonance elastography (MRE) in screening patients for hepatic fibrosis, cirrhosis, and hepatocellular carcinoma after the Fontan operation. PATIENTS AND METHODS Hepatic MRE was performed in conjunction with cardiac magnetic resonance imaging in patients who had undergone a Fontan operation between 2010 and 2014. Liver stiffness was calculated using previously reported techniques. Comparisons to available clinical, laboratory, imaging, and histopathologic data were made. RESULTS Overall, 50 patients at a median age of 25 years (range, 21-33 years) who had undergone a Fontan operation were evaluated. The median interval between Fontan operation and MRE was 22 years (range, 16-26 years). The mean liver stiffness values were increased: 5.5 ± 1.4 kPa relative to normal participants. Liver stiffness directly correlated with liver biopsy-derived total fibrosis score, time since operation, mean Fontan pressure, γ-glutamyltransferase level, Model for End-Stage Liver Disease score, creatinine level, and pulmonary vascular resistance index. Liver stiffness was inversely correlated with cardiac index. All 3 participants with hepatic nodules exhibiting decreased contrast uptake on delayed postcontrast imaging and increased nodule stiffness had biopsy-proven hepatocellular carcinoma. CONCLUSION The association between hepatic stiffness and fibrosis scores, Model for End-Stage Liver Disease scores, and γ-glutamyltransferase level suggests that MRE may be useful in detecting (and possibly quantifying) hepatic cirrhosis in patients after the Fontan operation. The correlation between stiffness and post-Fontan time interval, mean Fontan pressure, pulmonary vascular resistance index, and reduced cardiac index suggests a role for long-term hepatic congestion in creating these hepatic abnormalities. Magnetic resonance elastography was useful in detecting abnormal nodules ultimately diagnosed as hepatocellular carcinoma. The relationship between stiffness with advanced fibrosis and hepatocellular carcinoma provides a strong argument for additional study and broader application of MRE in these patients.

Role in Tumor Growth of a Glycogen Debranching Enzyme Lost in Glycogen Storage Disease

BACKGROUND Bladder cancer is the most common malignancy of the urinary system, yet our molecular understanding of this disease is incomplete, hampering therapeutic advances. METHODS Here we used a genome-wide functional short-hairpin RNA (shRNA) screen to identify suppressors of in vivo bladder tumor xenograft growth (n = 50) using bladder cancer UMUC3 cells. Next-generation sequencing was used to identify the most frequently occurring shRNAs in tumors. Genes so identified were studied in 561 patients with bladder cancer for their association with stratification of clinical outcome by Kaplan-Meier analysis. The best prognostic marker was studied to determine its mechanism in tumor suppression using anchorage-dependent and -independent growth, xenograft (n = 20), and metabolomic assays. Statistical significance was determined using two-sided Student t test and repeated-measures statistical analysis. RESULTS We identified the glycogen debranching enzyme AGL as a prognostic indicator of patient survival (P = .04) and as a novel regulator of bladder cancer anchorage-dependent (P < .001), anchorage-independent (mean ± standard deviation, 180 ± 23.1 colonies vs 20±9.5 in control, P < .001), and xenograft growth (P < .001). Rescue experiments using catalytically dead AGL variants revealed that this effect is independent of AGL enzymatic functions. We demonstrated that reduced AGL enhances tumor growth by increasing glycine synthesis through increased expression of serine hydroxymethyltransferase 2. CONCLUSIONS Using an in vivo RNA interference screen, we discovered that AGL, a glycogen debranching enzyme, has a biologically and statistically significant role in suppressing human cancer growth.

Mammalian Target of Rapamycin Complex 2 (mTORC2) Is a Critical Determinant of Bladder Cancer Invasion

Bladder cancer is the fourth most common cause of cancer in males in the United States. Invasive behavior is a major determinant of prognosis. In this study, we identified mammalian target of rapamycin complex 2 (mTORC2) as a central regulator of bladder cancer cell migration and invasion. mTORC2 activity was assessed by the extent of phosphorylation of Ser473 in AKT and determined to be approximately 5-fold higher in specimens of invasive human bladder cancer as opposed to non-invasive human bladder cancer. The immortalized malignant bladder cell lines, UMUC-3, J82 and T24 demonstrated higher baseline mTORC2 activity relative to the benign bladder papilloma-derived cell line RT4 and the normal urothelial cell line HU1. The malignant bladder cancer cells also demonstrated increased migration in transwell and denudation assays, increased invasion of matrigel, and increased capacity to invade human bladder specimens. Gene silencing of rictor, a critical component of mTORC2, substantially inhibited bladder cancer cell migration and invasion. This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation. These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.

TFEB-VEGFA (6p21.1) co-amplified renal cell carcinoma: a distinct entity with potential implications for clinical management

A subset of renal cell carcinomas shows TFEB overexpression secondary to MALAT1-TFEB gene fusion. As alternate mechanisms of TFEB overexpression are likely to have the same effect, we sought to determine the frequency of amplification of TFEB and the adjacent VEGFA gene at 6p21.1. As patients with metastatic renal cell carcinomas are managed with anti-VEGF therapies, we retrospectively assessed therapeutic response in patients with amplified tumors. Amplification status was analyzed for 875 renal cell carcinomas from our institution, a consultative case and 794 cases from The Cancer Genome Atlas. Cases were classified as having low level (5–10 copies), and high-level amplification (>10 copies), and were further analyzed for adjacent oncogene copy number status (n=6; 3 single-nucleotide polymorphism genomic microarray, 3 The Cancer Genome Atlas) and structural rearrangements (n=1; mate-pair sequencing). These were then reviewed for histopathology, immunophenotype, and response to VEGF-targeted therapy on follow-up. In all, 10/875 (1.1%) institutional cases, 1 consultative case, and 3/794 (0.4%) of The Cancer Genome Atlas cases showed TFEB high-level amplification, while 14/875 (1.6%) cases showed TFEB low-level amplification. All cases had associated VEGFA amplification. This was confirmed with evaluation for copy number changes (n=6). The 6p21.1 high and low-level amplified tumors occurred in adults (mean age: 66), with over half being ≥pT3 (13/25, 52%), and most showed oncocytic, tubulopapillary features and high grade (≥grade 3: 20/22, 91%). These were aggressive tumors with metastasis and death from renal cell carcinoma in 11 (of 24, 46%) cases. Four patients received targeted therapy and had a mean survival of 31 months (range: 17–50) post nephrectomy. In summary, a group of aggressive renal cell carcinomas show genomic amplification of the 6p21.1 region including TFEB and VEGFA genes and share morphologic features. Additional studies are warranted to determine whether these patients respond to anti-VEGF therapy.

Outcome prediction for patients with renal cell carcinoma.

Outcome assessment for renal cell carcinoma is somewhat controversial. Despite numerous studies, a very limited variety of features have been recognized as having prognostic significance in clinical practice. In this review, tumor features considered to be of importance in outcome prediction for surgically treated patients with the 3 most commonly encountered morphotypes of renal cell carcinoma (clear cell, papillary, and chromophobe renal cell carcinoma) are evaluated. In particular, we have focused upon histologic subtype, sarcomatoid and rhabdoid differentiation, TNM staging, primary tumor size, tumor grade, and the presence of histologic coagulative tumor necrosis. We have also examined the importance of these prognostic features in a variety of postoperative or outcome prediction models developed by several institutions.

Sarcomatoid Urothelial Carcinoma of the Bladder: Analysis of 28 Cases With Emphasis on Clinicopathologic Features and Markers of Epithelial-to-Mesenchymal Transition

CONTEXT -Sarcomatoid urothelial carcinoma (UCa) is a rare but aggressive variant of bladder cancer that can show diagnostic challenges even using ancillary techniques. OBJECTIVE -To examine immunohistochemical markers in the context of sarcomatoid UCa, including those associated with epithelial-to-mesenchymal transition. DESIGN -Twenty-eight cases of sarcomatoid UCa were rereviewed. Clinical outcomes were obtained through database search. Immunohistochemistry for clinical and epithelial-to-mesenchymal transition markers was performed. RESULTS -All patients had biopsy-proven invasive UCa; 61% (17 of 28) had sarcomatoid UCa at initial diagnosis. A recognizable epithelial component(s) was present in 17 lesions. The sarcomatoid component accounted for 65% of the lesion (average), with heterologous elements present in 3 of 28 cases (11%). The morphologic spectrum of the sarcomatoid element included spindled not otherwise specified, myxoid, pseudoangiosarcomatous, and malignant fibrous histiocytoma-like undifferentiated features. The sarcomatoid component was immunoreactive for pancytokeratin (22 of 26; 85%), p63 (20 of 26; 77%), cytokeratin 903 (17 of 26; 65%), cytokeratin 7 (16 of 26; 62%), GATA3 (16 of 26; 62%), and cytokeratin 5/6 (16 of 26; 62%). STAT-6, CD31, CD34, and HMB45 were all nonreactive, whereas smooth muscle actin often showed at least focal immunoreactivity (22 of 26; 85%). Epithelial-to-mesenchymal transition markers were frequently expressed, including vimentin (26 of 26; 100%), FoxC2 (26 of 26; 100%), SNAIL (23 of 26; 88.5%), and ZEB1 (18 of 26; 69.2%). Follow-up was available for 24 patients (median, 7 months). Sixteen of 28 patients (57%) died of disease (overall mean survival, 9.1 months). The presence of myxoid or chordoid features was associated with reduced survival (P < .05). CONCLUSIONS -Sarcomatoid UCa is an aggressive form of UCa that frequently expresses epithelial-to-mesenchymal transition markers, suggesting a possible mechanism associated with aggressive tumor behavior.

Misidentification of Neosartorya pseudofischeri as Aspergillus fumigatus in a Lung Transplant Patient

ABSTRACT We present a case of disseminated Neosartorya pseudofischeri infection in a bilateral lung transplant patient with cystic fibrosis. The organism was originally misidentified from respiratory specimens as Aspergillus fumigatus using colonial and microscopic morphology. DNA sequencing subsequently identified the organism correctly as N. pseudofischeri.

Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2-Dependent Bladder Cancer Cell Migration and Invasion.

Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β-induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers.

Abstract 3652: Pegylated arginine deiminase (ADI-PEG20) as a potential therapeutic agent for rarer variants of bladder cancer that are deficient for argininosuccinate synthetase

Background: A previous study had identified microsatellite instability in the argininosuccinate synthetase (ASS1) gene locus in urothelial cancers (UCC), although the significance of this finding was unclear. ASS1 plays a critical role in the biosynthesis of arginine from citrulline and a deficiency of ASS1 leads to arginine auxotrophy. This can be further exacerbated by administration of the pegylated form of arginine deiminase (ADI-PEG20), which has been shown to carry low toxicity in phase I/II clinical trials. Thus, reduced levels of ASS1 can serve as an indicator for ADI-PEG20-responsive tumors. We therefore evaluated the expression of ASS1 in bladder cancer variants to explore the possibility of expanding therapeutic avenues for this population. Design: Formalin fixed, paraffin embedded samples of normal urothelium (n=19), conventional UCC (n=148), and other variants including the micropapillary variant of UCC (n=17), small cell carcinoma (n=19), squamous cell carcinoma (n=39) and adenocarcinoma (n=19), were used in the construction of tissue microarrays (TMAs). TMAs were immunostained for ASS1 and scored based on the intensity of stain (0-3+; high staining was considered 2-3+). The results were then correlated with various parameters such as age at diagnosis, sex, race and pathologic staging. Also, these results were correlated with ASS1 expression as determined by western blotting in multiple bladder cancer derived cell-lines. Results: Compared to normal urothelium where 68.4% of the cases showed high expression of ASS, conventional UCCs demonstrated lower levels of staining (41% of the non-metastatic subset; n=61 and 52.8% of the metastatic subset; n=87). Surprisingly, the paired lymph node metastases from the latter group showed high staining in only 31.5% (n=38) of the patients. High levels of ASS1 immunoreactivity were maintained in the micropapillary variant of UCC (76.4% of cases) and pure bladder adenocarcinoma (84.2%). In contrast, both pure small cell and squamous cell carcinoma of the bladder demonstrated marked reduction in ASS1 expression: only 26.3% of small cell carcinoma specimens and 10.2% of the squamous cell carcinoma specimens had high expression of ASS1, suggesting these two subtypes of bladder cancer could potentially respond to ADI-PEG20 therapy. We have also identified 6 bladder cancer cell-lines (including one derived from a squamous cell carcinoma) that are deficient for ASS1, and a primary cell-line which has high levels of ASS1 expression. These can serve as excellent tools for further in vitro studies. Conclusion: ADI-PEG20 represents a potentially exciting new therapeutic agent for metastatic conventional UCC, small cell carcinoma and squamous cell carcinoma. This is especially relevant as limited therapies are currently available for these less common forms of bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3652. doi:1538-7445.AM2012-3652

Argininosuccinate Synthetase 1 Loss in Invasive Bladder Cancer Regulates Survival through General Control Nonderepressible 2 Kinase–Mediated Eukaryotic Initiation Factor 2α Activity and Is Targetable by Pegylated Arginine Deiminase

Loss of argininosuccinate synthetase 1 (ASS1), a key enzyme for arginine synthesis, occurs in many cancers, making cells dependent on extracellular arginine and targetable by the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20). We evaluated ASS1 expression and effects of ASS1 loss in bladder cancer which, despite affecting >70,000 people in the United States annually, has limited therapies. ASS1 loss was identified in conventional and micropapillary urothelial carcinoma, small cell, and squamous cell carcinoma subtypes of invasive bladder cancer, as well as in T24, J82, and UM-UC-3 but not in 5637, RT112, and RT4 cell lines. ASS1-deficient cells showed preferential sensitivity to ADI-PEG 20, evidenced by decreased colony formation, reduced cell viability, and increased sub-G1 fractions. ADI-PEG 20 induced general control nonderepressible 2-dependent eukaryotic initiation factor 2α phosphorylation and activating transcription factor 4 and C/EBP homologous protein up-regulation, associated with caspase-independent apoptosis and autophagy. These effects were ablated with selective siRNA silencing of these proteins. ASS1 overexpression in UM-UC-3 or ASS1 silencing in RT112 cells reversed these effects. ADI-PEG 20 treatment of mice bearing contralateral flank UM-UC-3 and RT112 xenografts selectively arrested tumor growth in UM-UC-3 xenografts, which had reduced tumor size, reduced Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. This suggests that ASS1 loss occurs in invasive bladder cancer and is targetable by ADI-PEG 20.