Lelia Noble

Irreversible electroporation of the liver and liver hilum in swine

BACKGROUND Irreversible electroporation (IRE) is a novel, non-thermal form of ablation. We studied the safety and efficacy of IRE for the ablation of liver tissue around the liver hilum. We also studied the ability of triphenyltetrazolium chloride staining (TTC) to predict the zone of ablation after IRE. METHODS Eight swine underwent 20 ablations of the liver and liver hilum. Two monopolar probes were positioned 2 cm apart. IRE was performed using 90 pulses of 2500-3000 V/cm. IRE treatments were performed from 15 min to 14 days (n= 4) before sacrifice. RESULTS All animals survived. No major complications were encountered. Ablation width ranged from 2.27 to 4.45 cm and ablation height ranged from 1.5 to 1.8 cm. TTC staining demonstrated the zone of ablation in all animals. Hepatocyte necrosis occurs immediately adjacent to large central veins without evidence of heat sink. Bile ducts, portal veins and hepatic arteries appear to be more resistant to the effects of IRE. CONCLUSIONS IRE appears to be safe and effective for liver tissue ablation in the liver hilum. The portal structures appear more resistant to the effects of IRE. TTC staining can predict the zone of IRE ablation as early as 15 min after treatment.

Irreversible electroporation of the pancreas in swine: a pilot study.

BACKGROUND Irreversible electroporation (IRE) is a novel, non-thermal method of tissue ablation using short pulses of high-voltage DC current to ablate tissue. METHODS Irreversible electroporation of the pancreas was performed in four domestic female swine using two monopolar probes spaced 9-15 mm apart. Ninety pulses of 1500 V/cm were delivered for each ablation. RESULTS All animals survived for their designated times of 2 h (n = 1), 2 days (n = 1) and 14 days (n = 2), respectively. No procedure-related complications occurred. Three animals in which probes had been spaced at intervals of 10 ± 1 mm showed evidence of irreversible ablation, with ablation height ranging from < 10 mm to 21 mm and ablation width ranging from < 10 mm to 16 mm by gross appearance and triphenyltetrazolium chloride (TTC) staining. The only animal in which probes had been spaced at intervals of 15 mm did not show evidence of irreversible ablation at 2 weeks. This may be secondary to the wider probe spacing and relatively low voltage, which results in a mostly reversible form of electroporation without cell death. CONCLUSIONS Irreversible electroporation appears to be a safe method for pancreas tissue ablation. Staining with TTC can predict the zone of IRE ablation within 2 h of treatment.

Claudin Expression in High Grade Invasive Ductal Carcinoma of the Breast: Correlation with the Molecular Subtype

Claudin proteins are a major component of the tight junctions. Dysregulation of claudin protein expression has been described in a number of malignancies. Gene expression profiling has stratified breast cancers into distinct molecular subtypes: luminal, HER2 positive (HER2+), and basal-like. Recently, a novel claudin-low molecular subtype has been described. In this study, we correlated the expression patterns of claudins with the molecular subtypes of breast cancer. On the basis of immunohistochemical expression, 226 grade 3 invasive ductal carcinomas were stratified into 65 luminal (estrogen receptor positive (ER+)), 65 HER2+, 86 basal-like, including 14 metaplastic carcinomas (ER−, HER2−, CK5/6, and/or epidermal growth factor receptor positive), and 10 unclassified. Tissue microarrays were analyzed for the expression of claudins 1, 3, 4, 7, and 8 by immunohistochemistry and scored semiquantitatively. High levels of expression were detected in 17% of all cases for claudin 1, 32% claudin 3, 41% claudin 4, 44% claudin 7, and 40% claudin 8. Luminal cancers exhibited increased claudins 7 and 8; basal-like tumors demonstrated increased expression of claudins 1 and 4. Low expression of all five claudins was detected in 30 of 226 cases (13%) and this group was designated ‘claudin-low’. The majority of the claudin-low subgroup were basal-like cancers (23 of 30, 77%). In contrast, only 1 of 30 (3%) claudin-low tumors was of the luminal phenotype and 6 of 30 cases (20%) were HER2+ (P<0.001). Within the basal-like subgroup, 64% of the metaplastic and 19% of the non-metaplastic tumors were claudin-low. The claudin-low group was strongly associated with disease recurrence (P=0.0093). In conclusion, this study is the first to examine comprehensively the differential expression of claudins 1, 3, 4, 7, and 8 in the molecular subtypes of high-grade breast cancer. Claudin-low subtype is a frequent phenomenon in metaplastic and basal-like breast cancer and appears to be a strong predictor of disease recurrence.

The diagnostic and prognostic utility of claudin expression in renal cell neoplasms

This study evaluated the expression patterns of claudins 1, 3, 4, 7, and 8 in human renal cell carcinomas and oncocytomas and correlated expression with patient prognosis. Tissue microarrays were created from paraffin-embedded tissue samples from 141 patients with renal cell carcinomas or oncocytoma (90 clear cell, 22 papillary, 17 chromophobe renal cell carcinomas, and 12 oncocytomas). The staining pattern for claudins 3, 4, 7, and 8 was membranous and/or cytoplasmic, whereas claudin 1 was predominantly membranous in both nonneoplastic renal tissue and tumors. Negative to weak claudin 3 staining was predominantly detected in Fuhrmans grade 1 and 2 clear cell renal cell carcinomas (78%; P=0.016), suggesting that upregulation of claudin 3 potentially occurs concomitantly with increasing grade of clear cell renal cell carcinomas. In addition, Kaplan–Meier univariate analysis showed a significant inverse correlation between moderate to strong claudin 3 and 4 expression with overall survival in clear cell renal cell carcinomas (P=0.038 and P=0.031). Moderate to strong claudin 7 expression was significantly more common in chromophobe renal cell carcinomas (94%) than in oncocytomas (55%; P=0.041). Claudin 8 staining was moderate to strong in 92% of oncocytomas, which differentiated them from papillary and clear cell renal cell carcinomas (14 and 12%; both P<0.0001). Only negative to weak claudin 8 staining was detected in all chromophobe renal cell carcinomas, whereas there were no claudin 8 negative oncocytomas and 8% exhibited a weak staining pattern (P<0.0001). Due to their distinctive expression patterns, claudins 7 and 8 can be used as useful immunohistochemical markers for the separation of chromophobe renal cell carcinomas from oncocytomas, whereas claudins 3 and 4 may serve as indicators of prognosis in clear cell renal cell carcinomas.

DIFFERENTIATING THE UNDIFFERENTIATED: IMMUNOHISTOCHEMICAL PROFILE OF MEDULLARY CARCINOMA OF THE COLON WITH AN EMPHASIS ON INTESTINAL DIFFERENTIATION

Undifferentiated or medullary carcinoma is characterized by its distinct histologic appearance and relatively better prognosis compared to poorly differentiated colonic carcinoma. These 2 entities may be difficult to differentiate by light microscopy alone. Only limited immunohistochemical studies investigating medullary carcinoma have been reported. These studies suggest a loss of intestinal differentiation, exemplified by a high percentage of CDX2 negativity. Our aim was to further characterize the immunohistochemical profile of medullary carcinoma, with particular emphasis on intestinal markers. Paraffin blocks from 16 cases of medullary carcinoma and 33 cases of poorly differentiated colonic carcinoma were retrieved, and tissue microarrays were constructed and stained with an immunohistochemical panel including CDX2, CK7, CK20, p53, intestinal trefoil factor 3, chromogranin, synaptophysin, MLH-1, MUC-1, MUC-2, and calretinin. A significantly higher proportion of medullary carcinomas, as opposed to poorly differentiated colonic carcinomas, showed loss of staining for MLH-1 and for the intestinal transcription factor CDX2, in accordance with previous studies. MLH-1 staining was present in only 21% of medullary carcinoma cases compared with 60% of the poorly differentiated colonic carcinoma cases (P = .02), whereas CDX2 was positive in 19% of medullary carcinomas and 55% of poorly differentiated colonic carcinomas (P = .03). Interestingly, calretinin staining was strongly positive in 73% of medullary carcinomas compared to only 12% of poorly differentiated colonic carcinomas (P < .0001). Evidence of intestinal differentiation by MUC-1, MUC-2, and TFF-3 staining was seen in 67%, 60%, and 53% of the medullary carcinomas, respectively. These 3 markers were frequently positive in many of the CDX2-negative medullary carcinoma cases. Medullary carcinoma of the colon retains a significant degree of intestinal differentiation as evidenced by its high percentage of staining for MUC-1, MUC-2, and TFF-3. Calretinin, MLH-1, and CDX2 may help to differentiate medullary carcinoma from poorly differentiated colonic carcinoma of the colon.

Expression of bile acid receptor TGR5 in gastric adenocarcinoma

Bile reflux is a risk factor in the development of intestinal metaplasia in the stomach and is believed to function as an initiator of gastric carcinogenesis. However, whether the G protein-coupled bile acid receptor TGR5 is expressed in this tumor is not known. In this study, we determined the expression of TGR5 in gastric adenocarcinoma and examined the role of TGR5 in cell proliferation. Strong TGR5 staining was present in 12% of cases of intestinal metaplasia but in no cases of normal gastric epithelium (P < 0.01). Moderate to strong TGR5 membranous and cytoplasmic staining was present in 52% of the intestinal but in only 25% of the diffuse subtype of adenocarcinomas (P < 0.001). Kaplan-Meier univariate survival analysis revealed that moderate to strong TGR5 staining was associated with decreased patient survival (P < 0.05). Treatment with taurodeoxycholic acid (TDCA, a bile acid) significantly increased thymidine incorporation in the AGS gastric adenocarcinoma cell line, suggesting that bile acids may increase cell proliferation. This increase was significantly decreased by knockdown of TGR5 with TGR5 small-interfering RNA (siRNA). In addition, overexpression of TGR5 significantly enhanced TDCA-induced increases in thymidine incorporation. TGR5 is coupled with G(q)α and Gα(i-3) proteins. TDCA-induced increase in thymidine incorporation was significantly decreased by knockdown of G(q)α and Gα(i-3) with their siRNAs. We conclude that TGR5 is overexpressed in most gastric intestinal-type adenocarcinomas, and moderate to strong TGR5 staining is associated with decreased patient survival in all gastric adenocarcinomas. Bile acids increase cell proliferation via activation of TGR5 receptors and G(q)α and Gα(i-3) proteins.

Expression microarray analysis identifies novel epithelial-derived protein markers in eosinophilic esophagitis.

Gene expression studies in eosinophilic esophagitis support an immune-mediated etiology associated with differential regulation of inflammatory and epithelial-derived genes. We aimed to further characterize epithelial gene expression alterations in eosinophilic esophagitis and to explore the use of immunohistochemistry to identify these alterations. Esophageal biopsies from pediatric patients with eosinophilic esophagitis before and after therapy with topical steroids (N=7) were screened by gene expression microarray and results were validated by RT-PCR. A larger group of eosinophilic esophagitis patients (N=42) was then used to evaluate protein expression by immunohistochemistry compared with reflux patients (N=15) and normal controls (N=17). Microarray and RT-PCR studies identified overexpression of ALOX15 and tumor necrosis factor alpha-induced factor 6 (TNFAIP6) and underexpression of filaggrin (FLG), SLURP1 and cysteine-rich secretory protein 3 (CRISP3) in eosinophilic esophagitis. Immunohistochemistry for ALOX15 was positive in 95% of eosinophilic esophagitis and negative in all controls, all eosinophilic esophagitis after therapy and all reflux biopsies (P<0.001). TNFAIP6 was positive in 88% of eosinophilic esophagitis samples versus 47% of controls, 29% of eosinophilic esophagitis after therapy and 40% of reflux samples (P=0.002). Overexpression of both ALOX15 and TNFAIP6 directly correlated with the degree of eosinophilic infiltration. FLG was positive in 88% of controls and 100% of reflux biopsies, but negative in all eosinophilic esophagitis samples, and its expression was regained in 86% of eosinophilic esophagitis after therapy patients (P<0.001). SLURP1 expression was positive in all controls and reflux samples, but only positive in 5% of eosinophilic esophagitis and was re-expressed to 100% positivity in eosinophilic esophagitis after therapy patients (P<0.001). The majority of controls (89%) and reflux biopsies (100%) were positive for CRISP3 while eosinophilic esophagitis before therapy were positive in 14% of samples (P<0.001) with partial recovery after treatment (43%, P=0.105). This study identified five epithelial-derived markers differentially expressed in eosinophilic esophagitis easily detectable by immunohistochemistry with potential diagnostic utility.

GCDFP-15 positive and TTF-1 negative primary lung neoplasms: a tissue microarray study of 381 primary lung tumors.

Gross cystic disease fluid protein (GCDFP-15) is currently used as an immunohistochemical marker of breast cancer, whereas thyroid transcription factor (TTF-1) is commonly used as a marker of primary lung neoplasms. Traditionally, a GCDFP-15+/TTF-1− immunohistochemical profile in lung tumors has been considered as highly suggestive of metastatic carcinoma of the breast. Here, we investigated the expression of GCDFP-15 and TTF-1 on a tissue microarray consisting of 381 primary lung carcinomas. GCDFP-15 was expressed in normal bronchial submucosal glands and bronchial epithelium, which were negative for TTF-1. Seventeen tumors (4.5%) were positive for GCDFP-15, including 11 of 186 (5.9%) adenocarcinomas, 1 of 97 (1%) squamous cell carcinomas, 1 of 23 (4.3%) carcinoid tumors, 2 of 47 (4.3%) large cell carcinomas, and 2 of 17 (11.8%) adenosquamous carcinomas. Of the 11 GCDFP-15 positive adenocarcinomas, 10 (91%) were TTF-1 negative. On whole sections, about half (55%) of GCDFP-15 positive cases were negative and one-third (33%) revealed focal TTF1 staining in areas distinct from the tumor regions that expressed GCDFP-15. All GCDFP-15 positive tumors were negative for mammaglobin, estrogen receptor, and progesterone receptor. Our study confirms that a small subset of primary lung adenocarcinomas exhibits a GCDFP-15 positive phenotype. Expression of TTF-1 in this group is not uniform and frequently negative in small specimens. Thus a GCDFP-15 positive/TTF-1 negative phenotype may not be indicative of metastatic breast carcinoma in every case. It is critical that pathologists be aware of this phenotypic subset of lung adenocarcinomas, especially when faced with small tissue or cytologic samples.

Expression of the intestinal biomarkers Guanylyl cyclase C and CDX2 in poorly differentiated colorectal carcinomas

Guanylyl cyclase C, a receptor for bacterial diarrheagenic enterotoxins, is expressed selectively by intestinal epithelium and is an endogenous downstream target of CDX2. The expression of Guanylyl cyclase C is preserved throughout the adenoma/carcinoma sequence in the colorectum. Detection of Guanylyl cyclase C expression by reverse transcriptase-polymerase chain reaction is currently being validated as a technique to identify occult lymph node metastases in patients with colorectal cancer and for circulating cells in the blood for postoperative surveillance. Although Guanylyl cyclase C is widely expressed by well-differentiated colorectal cancer, its expression in poorly differentiated colorectal cancer has not been evaluated. A tissue microarray was created from 69 archival specimens including 44 poorly differentiated, 15 undifferentiated or medullary, and 10 signet ring cell colorectal carcinomas. Matched normal colonic mucosa was used as a positive control. Immunohistochemical staining for Guanylyl cyclase C and CDX2 was evaluated as positive or negative based on at least a 10% extent of staining. Of the 69 tumor samples, 75%, 47%, and 90% of the poorly differentiated, medullary, and signet ring cell tumors were positive for Guanylyl cyclase C and 75%, 40% and 90% of these subsets were positive for CDX2, respectively. There was excellent correlation between Guanylyl cyclase C and CDX2 expression on a case-per-case basis (P < .0001). There was also a statistically significant difference in the Guanylyl cyclase C staining pattern between medullary carcinomas and poorly differentiated, not otherwise specified (P = .05). Immunopositivity for Guanylyl cyclase C was greater than 95% in a separately stained microarray series of well/moderately differentiated colorectal carcinomas. In conclusion, Guanylyl cyclase C expression is lost in a quarter of poorly differentiated and half of undifferentiated colorectal carcinomas. Therefore, the utility of Guanylyl cyclase C expression as a diagnostic marker for colorectal carcinoma may be questionable in poorly differentiated colorectal neoplasms.

Mineralocorticoid Receptor and 11??-Hydroxysteroid Dehydrogenase Type II Expression in Renal Cell Neoplasms: A Tissue Microarray and Quantitative RT-PCR Study

The kidney is an important target for mineralocorticoids. Aldosterone, the major endogenously secreted mineralocorticoid, acts by binding to mineralocorticoid receptor (MR) in the distal renal tubule. The enzyme 11β-hydroxysteroid dehydrogenase type II (11β-HSD2) prevents the binding of glucocorticoids to the MR by inactivating cortisol to cortisone. Our goal was to determine whether MR and 11β-HSD2 expression could be used to characterize the major types of renal cell neoplasms. Using immunohistochemistry we analyzed tissue microarray specimens from 132 patients with renal cell neoplasms, stratified into 84 clear cell renal cell carcinomas (CRCC), including 9 cases clear cell carcinoma with predominantly granular cytoplasm; 14 papillary RCC (PRCC); 20 chromophobe RCC (CHRCC); and 14 oncocytomas (OCs). MR and 11β-HSD2 expression were also quantitated by real-time reverse transcription-polymerase chain reaction. Expression of both MR and 11-βHSD2 was detected in the distal nephrons of normal kidneys. The CHRCC group stained for 11-βHSD2 in a membranous and cytoplasmic pattern whereas diffuse cytoplasmic reactivity was seen in OCs. MR and 11β-HSD2 were coexpressed in most of CHRCC (90% and 95%) and oncocytomas (93% and 100%). No MR staining was detected in CRCC, including clear cell carcinoma with predominantly granular cytoplasm, or in PRCC. Only 2 cases of CRCC (2.6%) showed focal positivity for 11β-HSD2, whereas all PRCCs were negative. CHRCC and OC demonstrated significantly higher levels of MR and 11β-HSD2 expression than CRCC and PRCC by real-time polymerase chain reaction. Moreover, CHRCC showed higher expression of MR and 11β-HSD2, as compared with OC. Our study indicates MR and 11β-HSD2 are both sensitive and specific markers of the distal nephron and its related neoplasms (CHRCC and OC). Additionally, the staining pattern and the level of MR and 11β-HSD2 expression seems to be useful in the distinction of CHRCC from OC. MR and 11β-HSD2 should be considered in the immunohistochemical panel to more accurately subtype renal cell tumors.