Shaolei Lu

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.

MicroRNA profiling in mucosal biopsies of eosinophilic esophagitis patients pre and post treatment with steroids and relationship with mRNA targets.

Background The characterization of miRNAs and their target mRNAs involved in regulation of the immune process is an area of intense research and relatively little is known governing these processes in allergic inflammation. Here we present novel findings defining the miRNA and mRNA transcriptome in eosinophilic esophagitis (EoE), an increasing recognized allergic disorder. Methods Esophageal epithelial miRNA and mRNA from five paired biopsies pre- and post-treatment with glucocorticosteroids were profiled using Taqman and Affymetrix arrays. Validation was performed on additional paired biopsies, untreated EoE specimens and normal controls. Differentially regulated miRNAs and mRNAs were generated, within which miRNA-mRNA target pairs with high predicted confidence were identified. Results Compared to the post-glucocorticoid treated esophageal mucosa, of all the 377 miRNA sequences examined, 32 miRNAs were significantly upregulated and four downregulated in the pre-treated biopsies. MiR-214 was the most upregulated (150 fold) and miR-146b-5b, 146a, 145, 142-3p and 21 were upregulated by at least 10 fold. Out of 12 miRNAs chosen for validation by qRT-PCR, five (miR-214, 146b-5p, 146a, 142-3p and 21) were confirmed and 11 shared the same trend. When the expression of the 12 miRNAs in the EoE mucosa was compared to unrelated normal mucosa, six (miR-214, 146b-5p, 146a, 21, 203, and 489) showed similar significant changes as in the paired samples and 10 of them shared the same trend. In the same five pairs of samples used to profile miRNA, 311 mRNAs were down-regulated and 35 were up-regulated in pre-treated EoE mucosa. Among them, 164 mRNAs were identified as potential targets of differentially regulated miRNAs. Further analysis revealed that immune-related genes, targeted and non-targeted by miRNAs, were among the most important genes involved in the pathogenesis of EoE. Conclusions Our findings add to the accumulating body of data defining a regulatory role for miRNA in immune and allergic processes.

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.

Medullary carcinoma of the colon: a distinct morphology reveals a distinctive immunoregulatory microenvironment

Medullary carcinoma of the colon is a unique histologic subtype of microsatellite unstable colorectal carcinoma but little is known regarding its tumor-immunoregulatory microenvironment. The aims of this study were to characterize the immune environment of medullary carcinoma and compare it with other microsatellite unstable and microsatellite stable colorectal carcinomas. An initial gene expression microarray analysis of six cases of medullary carcinoma was used to detect potentially differentially expressed genes. We extended this analysis utilizing genomic data from the Cancer Genome Atlas to compare eight cases of medullary carcinoma with other microsatellite unstable and stable carcinomas. Finally, we evaluated expression of key immune pathway proteins and lymphocyte subsets via immunohistochemistry of a large group of medullary carcinomas (n=105) and compared these findings with three other groups: poorly differentiated, microsatellite unstable well-differentiated and microsatellite stable well-differentiated carcinomas. Microarray and the Cancer Genome Atlas data analysis identified significant upregulation of several immunoregulatory genes induced by IFNγ including IDO-1, WARS (tRNA(trp)), GBP1, GBP4, GBP5, PDCD1 (PD-1), and CD274 (PD-L1) in medullary carcinoma compared with other microsatellite unstable and microsatellite stable tumors. By immunohistochemistry, IDO-1 was expressed in 64% of medullary carcinomas compared with 19% (9/47) of poorly differentiated carcinomas, 14% (3/22) of microsatellite unstable, and 7% (2/30) of the microsatellite stable well-differentiated carcinomas (P<0.0001). tRNA(trp) was overexpressed in 81% (84/104) of medullary carcinomas, 19% (9/47) of poorly differentiated, 32% (7/22) of microsatellite unstable, and 3% (1/30) of microsatellite stable well-differentiated carcinomas (P<0.0001). Medullary carcinoma had higher mean CD8+ and PD-L1+ tumor-infiltrating lymphocytes compared with all other groups (P<0.0001). This study demonstrates overexpression of several immunoregulatory genes in microsatellite unstable colorectal carcinomas and that expression of these genes and proteins is more prevalent in the medullary carcinoma subtype, which may be of use both diagnostically and therapeutically.

RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients

BackgroundA major obstacle in treating colorectal cancer (CRC) is the acquired resistance to chemotherapeutic agents. An important protein in the regulation of cancer cell death and clinical outcome is Raf kinase inhibitor protein (RKIP). In contrast, activated signal transducer and activator of transcription 3 (STAT3) is a protein that promotes tumor cell survival by inhibiting apoptosis and has an important role in cancer progression in many of cancer types. The aim of this study was to evaluate the regulation of RKIP and STAT3 after treatment with clinically relevant chemotherapeutic agents (camptothecin (CPT) and oxaliplatin (OXP)) and the cytokine interleukin-6 (IL-6) in HCT116 colon cancer cells as well as evaluate the association between RKIP and STAT3 with clinical outcome of Stage II colon cancer patients.MethodsHCT-116 colon cancer cells were treated with CPT, OXP, and IL-6 separately or in combination in a time and dose-dependent manner and examined for phosphorylated and non-phosphorylated RKIP and STAT3 via Western blot analysis. STAT3 transcriptional activity was measured via a luciferase reporter assay in HCT116 cells treated with CPT, IL-6 or transfected with JAK 1, 2 separately or in combination. We extended these observations and determined STAT3 and RKIP/ pRKIP in tumor microarrays (TMA) in stage II colon cancer patients.ResultsWe demonstrate IL-6-mediated activation of STAT3 occurs in conjunction with the phosphorylation of RKIP in vitro in human colon cancer cells. OXP and CPT block IL-6 mediated STAT3 activation and RKIP phosphorylation via the inhibition of the interaction of STAT3 with gp130. We determined that STAT3 and nuclear pRKIP are significantly associated with poor patient prognosis in stage II colon cancer patients.ConclusionsIn the analysis of tumor samples from stage II colon cancer patients and the human colon carcinoma cell line HCT116, pRKIP and STAT3, 2 proteins potentially involved in the resistance to conventional treatments were detected. The phosphorylation of pRKIP and STAT3 are induced by the cytokine IL-6 and suppressed by the chemotherapeutic drugs CPT and OXP. Therefore, these results suggest that STAT3 and pRKIP may serve as prognostic biomarkers in stage II colon cancer patients and may improve chemotherapy.

Correlation of ALOX15 expression with eosinophilic or reflux esophagitis in a cohort of pediatric patients with esophageal eosinophilia

The differential diagnosis between eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) is often challenging. We recently showed that the ALOX15 protein is expressed in 95% of esophageal biopsies from patients with a definitive diagnosis of EoE. Here we correlated ALOX15 expression with the clinical classification of EoE or GERD in a cohort of consecutive pediatric patients (n = 62) with at least 1 esophageal biopsy containing at least 15 eosinophils per high-power field (eos/HPF). The patients were categorized into the following groups: (1) at least 15 eos/HPF in the distal esophagus only (n = 24), (2) at least 15 eos/HPF in the proximal esophagus only (n = 5), and (3) at least 15 eos/HPF in the distal and proximal biopsies (n = 33). Control groups included patients with GERD with biopsies containing 6 to 15 eos/HPF (n = 9), patients with GERD with 5 eos/HPF or less (n = 15), patients with candida esophagitis (n = 15), and patients with normal biopsies (n = 15). ALOX15 was positive in 90.5% of patients with EoE (13/16 in group 1, 4/4 in group 2, 31/33 in group 3) versus 44% of patients with GERD (4/8 in group 1, 0/1 in group 2, and 0/0 in group 3), 2 of 9 (22%) of patients with 6 to 15 eos/HPF, and was negative in all patients with GERD with biopsies containing 5 eos/HPF or less, all patients with candida esophagitis, and all normal controls. In conclusion, ALOX15 is a sensitive marker of EoE; however, subpopulations of patients with GERD with >5 eos/HPF also express ALOX15. Positive ALOX15 expression is more prevalent in EoE than in GERD and may prove to be a useful diagnostic marker in patients with discrepant biopsy findings between the proximal and distal esophagus.

Calretinin expression in high-grade invasive ductal carcinoma of the breast is associated with basal-like subtype and unfavorable prognosis.

Calretinin, a calcium-binding protein, is a widely used marker for mesothelial differentiation. There is accumulating evidence of calretinin expression in epithelial and mesenchymal malignancies, as well. The objectives of this study were to (1) further delineate the expression of calretinin in grade 3 breast carcinomas in the context of molecular subtypes and (2) identify the impact of calretinin expression on overall and disease-free survival. On the basis of immunohistochemical expression of estrogen receptor, progesterone receptor, human epidermal growth factor receptor-2 (HER2), cytokeratin 5/6, and epidermal growth factor receptor, 214 grade 3 invasive ductal carcinomas were stratified into 36 luminal A, 63 luminal B, 24 HER2 positive, 81 basal-like (including 13 metaplastic carcinomas), and 10 unclassified. Tissue microarrays were analyzed for immunohistochemical expression of calretinin. High-level calretinin expression was identified in a significant proportion of basal-like (54.3%), HER2 (33.3%), and unclassified (30%) tumors. In contrast, luminal A and B subtypes demonstrated high-level calretinin expression in only 11.1% and 12.7%, respectively (P < .0001). Within the basal-like group, 38.5% of the metaplastic carcinomas demonstrated high-level expression, associated predominantly with the epithelial component and squamous metaplasia. High-level calretinin expression was strongly associated with decreased overall survival in the entire cohort of grade 3 cancer (P = .0096) and in the basal-like group (P = .039). Multivariate analysis revealed that both tumor stage and high-level calretinin expression were independent predictors of overall survival (P = .0002 and P = .0023, respectively). In conclusion, high-level calretinin expression is most common in grade 3 tumors with a basal-like phenotype and is associated with poor overall survival.

Intra-operative microwave ablation of liver malignancies with tumour permittivity feedback control: a prospective ablate and resect study

BACKGROUND Tumour permittivity feedback control is a novel method for microwave ablation (MWA) that theoretically allows for larger, more predictable ablations. This prospective case series evaluates the feasibility and efficacy of MWA of liver malignancies using a device with tumour permittivity feedback control. METHODS Ten consecutive patients initially determined to be candidates for surgical resection of a liver malignancy underwent intra-operative MWA with tumour permittivity feedback control followed by a surgical resection. A 14-gauge Medwaves microwave antenna was used to deliver a single treatment according to the manufacturers recommendations. Tumours were assessed grossly as well as by haematoxylin and eosin (H&E) and tetrazolium chloride staining. The primary end point was per cent tumour necrosis. RESULTS The median maximum ablation diameter measured was 4.1 cm (range 3.0-6.8). The median ablation volume was 8.7 cm(3) (range 4.84-17.55). Six of the 10 tumours demonstrated a pathological complete response (CR). Six of seven tumours ≤ 3 cm demonstrated a pathological CR. Zero of the three tumours ≥ 3 cm had a pathological CR, but all had ≥ 50% tumour necrosis. All patients survived and there were no ablation-related morbidities. DISCUSSION MWA of liver tumours with tumour permittivity feedback control is feasible and appears effective for the treatment of small (< 3 cm) liver tumours.

ARID1A alteration in aggressive urothelial carcinoma and variants of urothelial carcinoma

ARID1A mutation leads to loss of the products of this tumor-suppressor gene. Studies demonstrated ARID1A mutation in 20% of stage IV urothelial carcinomas (UCs) with worse prognosis. The expression of ARID1A in aggressive variants of UC is not studied properly. From 2000 to 2015, 81 variants of UC (29 micropapillary, 33 sarcomatoid, 31 small cell, 2 nested, and 3 plasmacytoid variants) were identified in the archives of Rhode Island Hospital. Immunohistochemistry for anti-ARID1A antibody (Sigma-Aldrich, St Louis, MO) was performed. The staining pattern was semiquantitatively scored, and results were analyzed by Fisher exact test (2 tailed) on contingency tables, survival curve, and log-rank test. Patients were predominantly male (78%) with mean age of 67.9 years. The plasmacytoid variant group occurred in younger ages (mean: 54 years). Half of the specimens contained concurrent conventional UCs. Normal urothelium invariably exhibited strong ARID1A nuclear staining. There was no difference in expression between upper and lower tracts. ARID1A expression was lower in the variants compared with conventional UCs (P<.0001). In micropapillary UCs, an inverse correlation between stage and ARID1A expression was noted, with significant correlation between ARID1A expression and overall survival (P=.0221). Sarcomatoid UCs and small cell CCs showed lower ARID1A expression compared with UCs that was not statistically significant, and neither showed any significant correlation with stage or overall survival. ARID1A expression is significantly decreased in higher stages of UC and its aggressive variants; therefore, ARID1A mutation appears to play an important role in the prognosis of UC and its aggressive variants. This finding may have therapeutic implications.

Cadherin 17 is a sensitive and specific marker for metanephric adenoma

Metanephric adenoma (MA) is a rare benign renal neoplasm that shares morphologic and immunophenotypic overlap with epithelial-predominant Wilms tumor (e-WT) and with the solid variant of papillary renal cell carcinoma (s-PRCC). Cadherin 17 (CDH17) is expressed primarily in the normal intestine and digestive tract tumors and has not been detected in tumors from other sites including the kidney. We investigated the diagnostic utility of CDH17 in differentiating between MA, e-WT, and s-PRCC. Immunohistochemical analysis for CDH17, CD57, AMACR, WT-1, and CDX2 was performed on 17 e-WTs, 15 s-PRCCs, and 21 MAs and assessed on the basis of a combined score of extent and intensity. Normal adult kidney parenchyma was negative for CDH17 staining. CDH17 was expressed in the late stages of fetal kidney development at the junction of the glomerular space and proximal nephron. The majority of MAs (81%) demonstrated membranous CDH17 immunoreactivity in all components (acinar, tubular, and papillary), whereas all cases of e-WTs and s-PRCCs were negative (P<0.0001). WT-1 was negative in s-PRCC and was positive in all cases of e-WT and MA. All MAs were strongly positive for CD57; however, this marker was also moderate to strongly positive in 6 (35%) e-WTs and 2 (13%) s-PRCCs. AMACR was strongly positive in all s-PRCCs, but moderate reactivity was seen in 3 (17%) e-WTs and 2 MAs (10%). CDH17 is a sensitive (81%) and highly specific (100%) marker for MA and should be considered in the immunohistochemistry panel for distinguishing MA from its mimics.