Uninvolved Tissue Research Articles

Abstract 3480: CD44 V6 and V9 isoforms act in resistance to Fas-mediated apoptosis in breast cancer initiating cells

Abstract Breast cancer initiating cells (BCIC) are CD44+/CD24 low/ESA+ cells derived from breast tumors that highly tumorigenic and resistant to apoptosis and to chemotherapy. To determine anti-apoptotic mediator (s) to both Fas death receptor signaling and chemotherapeutic agents induced apoptosis in BCIC, we detest the expression of V5, V6 and V9 of CD44 isoforms in BCIC derived from breast cancer cell lines MCF-7, MDA-MB-231, SUM159 and SUM1315, and assess their effects on both Fas death receptor signaling and chemotherapeutic agents induced apoptosis. Methods: 1). We measured relative expression of the various CD44 isoforms in parental and BCIC populations using quantitative real time-PCR. 2).We then determined which CD44 isoforms play a role in BCIC's resistance to apoptosis. SUM159 was chosen to use due to the overexpression of all CD44 isoforms. 3). After isolation of cells expressing the BCIC markers from SUM159 cells (CD44+/CD24 low/ESA+), we knocked down CD44 V5, V6 and V9 by siRNA in SUM159 BCIC cells and challenged them with increasing doses of the chemotherapeutic agent doxorubicin and FasL. 4).Tissue microarrays of human breast cancer (www.tissue-array.com) were also employed to assess association between the CD44 V9 expression and clinical parameters such as nodal metastasis, histological status, ER status, PgR status, HER2 status and survival in 40 cases of primary breast cancers. Results: 1).CD44 isoforms V5, V6 and V9 are differentially expressed in BCIC compared to their parental cell lines. V9 is markedly overexpressed in all 4 BCIC tested. 2). BCIC cells treated with siRNA targeting isoforms V5, V6 and V9 demonstrated no significant effect on doxorubicin IC50 when compared to controls. The existence of CD44 V9 and CD44 V6 in breast cancer cells confer resistance to apoptosis induced by FasL, knockdown of CD44 V6 resulted in 39% increase in caspase 3/7 activity when compared to controls while knockdown of CD44V9 caused a 49% increase. Further study showed that this is through the inhibition of the extrinsic apoptotic pathway by suppressing caspases-8 activation, thereby blocking downstream apoptotic events. 3). 40 cases of primary breast cancers provided assessable cores paired with corresponding uninvolved tissue from the same patient. The analysis showed a tendency between CD44V6 expression and HER2 status. We found that approximately 72.8% of HER2-positive patients express CD44V9 compared to 48.3% of HER2-negative patients expressing CD44V9. It did not reach statistically significance probably because of small size of samples. Conclusions: Loss of expression of the V6 and V9 but not V5 isoforms in SUM159 BCIC resulted in an increased sensitivity to apoptosis in response to the Fas L but not to the chemotherapeutic agent doxorubicin. These findings establish a role for CD44 V6 and V9 isoforms in aptoptotic resistance of BCIC and suggest that they may serve as targets for therapeutic intervention. 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 3480. doi:1538-7445.AM2012-3480

Abstract 1035: SETD2, a histone methyltransferase, is misregulated in advanced clear cell renal cell carcinoma (ccRCC)

Abstract Background: Although the von Hippel-Lindau (VHL) tumor suppressor is inactivated in more than 91% of clear cell renal cell carcinoma (ccRCC), VHL deletion in mice is insufficient for tumorigenesis, suggesting that additional mutations are required. Exome sequencing of early stage (I-III) ccRCC tumors identified missense and truncating mutations in SETD2, a histone H3 lysine 36 (H3K36) trimethyltransferase. However it is unclear whether these mutations alter histone modifications. We hypothesize that loss of SETD2 histone methyltransferase activity decreases H3K36 trimethylation (H3K36Me3) in ccRCC. Methods: To determine if loss of SETD2 protein expression alters H3K36Me3, SETD2 siRNA oligonucleotides were transfected into HEK293 (human embryonic kidney) cells and H3K36Me3 was analyzed by Western blot. In parallel, H3K36Me3 immunohistochemical (IHC) staining was quantitated in nephrectomy tissue with a mutant SETD2 genotype. To establish the frequency of SETD2 loss of heterozygosity (LOH), genomic DNA was isolated from 51 formalin-fixed paraffin-embedded ccRCC specimens and analyzed with Affymetrix GeneChip Mapping 250K Nsp single-nucleotide polymorphism (SNP) arrays. To determine if there are alterations of histone modifications in metastatic ccRCC, H3K36Me3 IHC staining was performed on tissue microarrays (TMAs) representing 28 paired ccRCC specimens with unaffected kidney parenchyma controls and 40 metastases (bone, brain and lungs). Results: SETD2 siRNA decreases H3K36Me3 (>50%) compared to scrambled controls suggesting that SETD2 is a non-redundant H3K36 methyltransferase. Nephrectomy specimens with SETD2 truncating mutations also have decreased H3K36Me3 (>50%) compared to matched uninvolved kidney tissue. Using SNP arrays, LOH at chromosome 3p (location of VHL and SETD2 genes) was detected in >90% of the tested ccRCC tumors. H3K36Me3 IHC staining was quantitated in TMAs representing advanced ccRCC specimens. IHC reveals a 27.4% and 52.0% decrease in H3K36Me3 positive nuclei in primary ccRCC and metastases, respectively, when compared to matched adjacent unaffected kidney tissue (P value <0.0001). Discussion: SETD2 siRNA transfected cell lines and patient-derived tissue with truncating SETD2 mutations have decreased H3K36Me3. In advanced ccRCC, LOH occurs in >90% of tested tumors. H3K36Me3 is decreased in primary ccRCC and even more in metastases, suggesting that SETD2 methyltransferase activity is progressively misregulated in RCC. VHL loss may cooperate with loss of SETD2 histone methyltransferase activity to promote tumorigenesis. Future studies will focus on the mechanism of how SETD2 contributes to tumorigenesis and on determining the prognostic and predictive impact of SETD2 phenotypic changes in patient-derived tissue. 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 1035. doi:1538-7445.AM2012-1035

Current status of near-total laryngectomy: review

Concurrent chemoradiation is currently the accepted 'standard of care' for locally advanced laryngeal and hypopharyngeal cancers. However, there is a subset of patients not suitable for chemoradiation, in whom primary surgery is the best option. Speech preservation is of prime importance in these patients. Near-total laryngectomy is a voice-preserving procedure which can be considered as an alternative to total laryngectomy for selected patients with lateralised, locally advanced cancers of the larynx and hypopharynx. Although these patients are left with a permanent tracheostomy, lung-powered speech is maintained by way of a dynamic shunt created from the uninvolved tissues of the larynx. Since its first description in the early 1980s, the procedure has been shown by various authors to be oncologically sound, with high success rates. Unfortunately, the procedure has not gained wide acceptance due to perceived fears of surgical complexity. In this review, we discuss the various issues related to the procedure and we review the relevant literature.

Learning from experience. Proposal of a refined definition and staging system for bisphosphonate‐related osteonecrosis of the jaw (BRONJ)

Learning from experience. Proposal of a refined definition and staging system for bisphosphonate‐related osteonecrosis of the jaw (BRONJ)

Reduced Expression of RNA Binding Protein CELF2, a Putative Tumor Suppressor Gene in Colon Cancer

Colon cancer is the third leading cause of cancer death in both men and women in the United States. Every year, 160000 cases of colorectal cancer are diagnosed, and 57000 patients die. CUGBP, Elav-like family member 2 (CELF2) is an RNA binding protein that modulates various posttranscriptional events including RNA splicing, shuttling, editing, stability and translation. Previous studies have demonstrated that CELF2 expression is low in colon cancer cells. Furthermore, ectopic overexpression of CELF2 induces cells to undergo death by mitotic catastrophe. Based on the above observations, we hypothesized that CELF2 expression might be reduced during neoplastic transformation of colon cells. Forty human colon cancer tissues along with 10 uninvolved normal colon tissues from cancer patients were utilized for immunohistochemical analysis of CELF2 expression. We have observed that CELF2 levels are reduced in colon tumor tissues when compared to the normal intestinal tissues. The data set suggests that RNA binding protein CELF2 could be a potential tumor suppressor protein. CELF2 was predominantly nuclear in normal cells, while the cancer tissues had diffused cytoplasmic staining. CELF2 expression is consistently reduced during neoplastic transformation suggesting that it might play a crucial role in tumor initiation and progression.

Treatment of Lung Metastases from Ewing Sarcoma with Stereotactic Body Radiotherapy rather than Whole-Lung Irradiation

ABSTRACT Advances in knowledge Lung metastases from Ewing sarcoma have traditionally been treated with whole-lung irradiation (WLI) and chemotherapy. WLI may unnecessarily treat uninvolved normal lung tissue and increase the risk of toxicity. Stereotactic body radiotherapy (SBRT) has proven to be a safe and effective treatment for primary lung cancer and lung metastases, but there is no significant published description of SBRT in lung metastases from Ewing sarcoma. Implications for patient care The patient presented in this case report had long-term survival from lung metastases of Ewing sarcoma treated with chemotherapy and SBRT. SBRT may prove to be safer and more effective than whole lung irradiation as a treatment of lung metastases in Ewing sarcoma. Summary statement Whether SBRT of lung metastases of Ewing sarcoma can improve outcomes and decrease toxicity compared to whole-lung irradiation, surgical resection or palliative chemotherapy merits further study. How to cite this article Rahn DA, Read W, Sandhu A. Treatment of Lung Metastases from Ewing Sarcoma with Stereotactic Body Radiotherapy rather than Whole-Lung Irradiation. J Postgrad Med Edu Res 2012;46(1):34-36.

Intra-operative re-excision of margins in breast-conserving surgery: How well are we able to judge tumour proximity?

s / International Journal of Surgery 10 (2012) S1–S52 S15 ABSTRACTS 1127: INTRA-OPERATIVE RE-EXCISION OF MARGINS IN BREASTCONSERVING SURGERY: HOW WELL ARE WE ABLE TO JUDGE TUMOUR PROXIMITY? Lorna Cook, Debasish Debnath, Zoe Lin, Raouf Daoud, Isabella Karat, Ian Laidlaw. Frimley Park Hospital, Frimley, UK Introduction: In breast-conserving surgery (BCS), specimen margin involvement with tumour often necessitates re-operation. For this reason, extra margins are often taken at the time of primary operation. The aim of this study was to determine how often such re-excisions are appropriate. Methods: The records of 100 consecutive patients undergoing BCS for cancer during 2011 were analysed. Data was collected on demographics, tumours characteristics, surgery performed and final histology. Results: All 100 patients were female, median age 56 years (range 33-83 years). 66% had intra-operative re-excision of margins. In 26/66 (40%), re-excision was the correct decision. 25 of the 34 patients who had no extramargins taken at all were managed appropriately with the remaining 9 patients having close/positive margins. Unnecessary re-excisions were performed in 40/66 patients. The correct intraoperative decision was therefore made in 51% of patients. Conclusion: The decision regarding intra-operative margin re-excision was appropriate in just over half of all cases and a second operation was avoided in 26%. However, in order to preserve as much uninvolved breast tissue as possible, further work should be done on methods to improve accuracy and to determine whether there are any patient, radiological or pathological predictors to guide intra-operative re-excision. 1181: FACTORS PREDICTING POSITIVE MARGIN STATUS IN BREAST CANCER PATIENTS UNDERGOING BREAST CONSERVING SURGERY Euan Harris , Andrew Lee . University of Dundee, Dundee, Tayside, UK; Abertawe Bro Morgannwg University Healthboard, Swansea, West

Adenomyoepithelioma A Rare Breast Tumor: Case Studies With Review Of The Literature

Introduction : Adenomyoepithelioma is a rare, benign proliferative tumor that can involve the breast tissue. The diagnostic appearance of the lesion can give rise to a difficult differential diagnosis with breast carcinoma. There is the potential for malignant evolution of this lesion. It usually presents as a solitary, unilateral, painless mass at the periphery of the breast. Accurate diagnosis and differentiation from more aggressive tumors is important. Materials and methods :In two cases of breast lumps, fine needle aspiration cytology (FNAC) was done and stained with H & E and Papanicolaou stains. The formalin-fixed tissue sections were stained with hematoxylin and eosin and in one case, immunohistochemistry was performed with smooth muscle actin (SMA).Case 1: A 30-year-old female presented with a firm lump in the upper quadrant of the right breast along with axillary lymphadenopathy since 6 months. On ultrasound it was reported as duct ectasia. FNAC was suggestive of proliferative breast disease with atypia (possibility of duct cell carcinoma). Modified radical mastectomy was done and sent for histopathology. The histopathological diagnosis was adenomyoepithelioma – breast (right), tubular type.Case 2: A 25-year-old female presented with a firm lump in the left breast, occupying all quadrants. On FNAC the aspirates were suggestive of fibroadenoma. Peroperatively it was found to be a cystic swelling occupying the whole breast. The mass was excised and sent for histopathology. The histopathological diagnosis was adenomyeoepithelioma with apocrine metaplasia and cystic degeneration – breast (left), tubular type.Results : Two cases of breast lump were managed surgically. Both the cases were females with the mean age being 27 years. Both cases were of adenomyoepithelioma (tubular type) with one case showing apocrine metaplasia and cystic change. Conclusion : Prognosis of patients with benign adenomyoepithelioma of the breast is usually good, but it has a potential for local recurrence, especially in the tubular and lobulated variants. Total surgical excision with an adequate margin of uninvolved breast tissue is therefore recommended.

Immunodetection of occult eosinophils in lung tissue biopsies may help predict survival in acute lung injury.

BackgroundAcute lung injury (ALI) is a serious respiratory disorder for which therapy is primarily supportive once infection is excluded. Surgical lung biopsy may rule out other diagnoses, but has not been generally useful for therapy decisions or prognosis in this setting. Importantly, tissue and peripheral blood eosinophilia, the hallmarks of steroid-responsive acute eosinophilic pneumonia, are not commonly linked with ALI. We hypothesized that occult eosinophilic pneumonia may explain better outcomes for some patients with ALI.MethodsImmunohistochemistry using a novel monoclonal antibody recognizing eosinophil peroxidase (EPX-mAb) was used to assess intrapulmonary eosinophil accumulation/degranulation. Lung biopsies from ALI patients (n = 20) were identified following review of a pathology database; 45% of which (i.e., 9/20) displayed classical diffuse alveolar damage (ALI-DAD). Controls were obtained from uninvolved tissue in patients undergoing lobectomy for lung cancer (n = 10). Serial biopsy sections were stained with hematoxylin and eosin (H&E) and subjected to EPX-mAb immunohistochemistry.ResultsEPX-mAb immunohistochemistry provided a >40-fold increased sensitivity to detect eosinophils in the lung relative to H&E stained sections. This increased sensitivity led to the identification of higher numbers of eosinophils in ALI patients compared with controls; differences using H&E staining alone were not significant. Clinical assessments showed that lung infiltrating eosinophil numbers were higher in ALI patients that survived hospitalization compared with non-survivors. A similar conclusion was reached quantifying eosinophil degranulation in each biopsy.ConclusionThe enhanced sensitivity of EPX-mAb immunohistochemistry uniquely identified eosinophil accumulation/degranulation in patients with ALI relative to controls. More importantly, this method was a prognostic indicator of patient survival. These observations suggest that EPX-mAb immunohistochemistry may represent a diagnostic biomarker identifying a subset of ALI patients with improved clinical outcomes.

Temporary Targeted Hemostasis to Facilitate Bloodless Partial Nephrectomy

To extend previous robotic-assisted techniques developed in the swine model to studies of laparoscopic and open partial nephrectomy conducted in pigs and calves, designed to encompass vessel diameters similar to those encountered in humans. Lumagel (Pluromed, Woburn, MA), a nontoxic polymer, can be administered intra-arterially under fluoroscopic guidance to obtain a bloodless operative field during partial nephrectomy while maintaining normal circulation to uninvolved renal tissue. A total of 10 animals (7 pigs and 3 calves) underwent flow interruption to the kidney, 2 with cross-clamping of the main renal artery, the remaining with Lumagel. Other than the first pig and calf, all the animals then underwent partial nephrectomy. Using Lumagel, targeted blood flow interruption was achieved and circulation to the uninvolved renal tissue was maintained. Hemostasis lasted for ≥30 minutes. The surgical resection time averaged 11 minutes (range 10-13) and 23.3 minutes (range 9-40) in the open and laparoscopic groups, respectively. The estimated blood loss was negligible, with the exception of 2 cases, 1 in which an error in angiographic assessment led to an unoccluded vessel near the resection site and a second case in which a guidewire was inadvertently passed through a vessel. The interval to complete flow return, as determined by direct visualization of the kidney and its corresponding angiogram, averaged 7 and 2.5 minutes for Lumagel and arterial clamping, respectively. Lumagel provides reliable and reproducible intraluminal blood flow interruption and flow restoration in both main and segmental renal arteries. By providing blood-free resection, the techniques described could facilitate partial nephrectomy without global renal ischemia.

Methylene Blue-enhanced Arthroscopic Resection of Dorsal Wrist Ganglions

The ganglion is the most common soft tissue mass of the hand and wrist. Over the past 10 to 15 years, there has been a growing interest in arthroscopic treatment of dorsal wrist ganglions. Proposed advantages of arthroscopy include greater motion (particularly wrist flexion), improved cosmesis, and potential to identify/treat other intra-articular pathology. Despite the documented clinical success of arthroscopic ganglion excision, limitations include inconsistent identification of the ganglion stalk. Our described technique offers a means by which to improve visualization of the ganglion stalk intra-articularly to produce a more effective and efficient arthroscopic ganglion excision. During the procedure, a small volume of methylene blue solution is injected into the cyst. Its communication with the joint is apparent arthroscopically, thus identifying the location of the stalk. With the ability to precisely identify the ganglion stalk using an injection of methylene blue, the surgeon can direct the arthroscopic debridement toward the appropriate pathologic tissue. Unnecessary debridement of uninvolved tissue can be avoided with the technique. This also allows for optimal portal placement and, in particular, indicates whether a midcarpal portal should be employed. This should result in fewer recurrences, decreased operative time, and less iatrogenic injury.

Postradiation Chronic Scalp Ulcer

For example, ionizing radiation, although beneficial for treating cancer, has negative effects on surrounding uninvolved tissues. Fragile and friable areas of damaged skin after radiation may lead to chronic, painful, and recalcitrant ulcers and, at times, bony changes. It may also result in more difficult healing after surgery and recurrence of the wound even with minor trauma. Despite improvements in radiation therapy techniques and equipment, chronic radiation ulcers are often resistant to conventional treatment modalities or advanced surgical options such as flap coverage or skin grafting and continue to be a therapeutic challenge. We present a case of a radiation ulcer treated using tissue engineered skin, discuss the pathophysiology of radiation-induced skin damage, and review the current management strategies.

Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer

We previously observed that the HERV type K (HERV-K) envelope (env) protein was expressed in the majority of human breast tumors from a U.S. cohort of women from Texas. We also made the preliminary observation that the expression of HERV-K env transcripts was associated with markers of disease progression. In this follow-up study, env protein expression was evaluated immunohistochemically in an additional 195 paraffin-embedded breast tumors from a second U.S. patient cohort (Baltimore, Maryland) and in 110 tumors from Chinese patients. Moreover, we compared env transcript expression between fresh-frozen normal and cancerous breast tissues. We observed that while env mRNA and protein expression was undetectable in normal breast tissue and in a subset of uninvolved normal-appearing tissue adjacent to the tumor epithelium, it was overexpressed in most tumors. Furthermore, env expression was associated with breast cancer progression. In Baltimore cohort women, HERV-K tumor positivity was significantly associated with disease stage and lymph node metastasis. In Chinese women, HERV-K env positivity was significantly associated with tumor size, TNM stage, and lymph node metastases, which is consistent with the observations in the U.S. cohort. We also found that Chinese breast cancer patients with a high expression of HERV-K had a decreased overall survival compared with patients who had either a moderate or low HERV-K expression in their tumors (P = 0.049, χ(2) log rank test). In conclusion, the HERV-K env gene is expressed in the majority of breast cancers from U.S. or Chinese women but not in normal breast tissue. High expression of HERV-K env protein in breast cancer patients is associated with markers of disease progression and poor disease outcome, indicating that HERV-K env protein is a novel candidate prognostic marker for breast cancer.

Clinical significance of histone deacetylases 1, 2, 3, and 7: HDAC2 is an independent predictor of survival in HCC

Histone deacetylases (HDAC) are responsible for the transcriptional control of genes through chromatin remodeling and control tumor suppressor genes. In several tumors, their expression has been linked to clinicopathological factors and patient survival. This study investigates HDACs 1, 2, 3, and 7 expressions in hepatocellular carcinoma (HCC) and their correlation with clinical data and patient survival. Tissue microarrays of 170 surgically resected primary HCCs and adjacent uninvolved tissue were evaluated immunohistochemically for the expression of HDACs 1, 2, 3, 7, and Ki-67 and were analyzed with respect to clinicopathological data and patient survival. HDACs 1, 2, 3, and Ki-67 were expressed significantly higher in cancer cells compared to normal tissue (HDAC1: p = 0.034, HDACs 2 and 3 and Ki-67: p < 0.001), while HDAC7 expression did not differ between HCC and non-cancerous liver tissue. In tumor tissue HDACs 1-3 expression levels showed high concordance with each other, Ki-67 and tumor grade (p < 0.001). High HDAC2 expression was associated with poor survival in low-grade and early-stage tumors (p < 0.05). The expression of the HDACs 1, 2, and 3 (but not HDAC7) isoenzymes correlates with clinicopathological factors, and HDAC2 expression has an impact on patient survival.

Pathway analysis of primary human non-small cell lung cancer (NSCLC).

10573 Background: We aimed to increase understanding of NSCLC pathogenesis through characterization of molecular and pathway signatures in primary lung adenocarcinoma (ADC) and squamous cell carcinoma (SSC) samples matched with syngeneic uninvolved normal lung tissue. Methods: Microarrary profiling of ADC and SSC matched with pathologically normal lung tissue (n=20 each) was conducted using the Affymetrix Human Gene 1.0 ST array. The differential expression profile of cancer vs syngeneic normal tissue was calculated for each patient and for combined samples using an unpaired t-test. Pathway analyses based on gene expression profiling were performed using Pathway Studio (Ariadne Genomics, Inc). Functional enrichment was performed using Fisher's exact test and Mann–Whitney test. Results: Transcriptional regulators E2F, CTGF and PDGF were implicated in lung cancer pathogenesis. SSC was characterized by involvement of EGF, IL1F8 and CX3CL1 pathways, while changes in RB1, mir-200 and EMP2 targets were specific for ADC. Cell cycle and metastatic processes were significantly affected in ADC and SCC. Cell cycle genes TOP2A, CCNB1, MELK, MAD2L1, SFN, CDC6, ASPM and TOPBP1 were significantly upregulated in SSC, opposed to just TOP2A in ADC. The biochemical signature pinpoints changes in purine and pyrimidine biosynthesis and energy production pathways, which were common in ADC and SSC. DNA repair genes RBBP8, PRKDC, SHFM1, and PCNA were significantly upregulated (2-fold or more; p<0.001) in SCC. All oncogenes in ADC, and most in SCC, were significantly downregulated, including FOS and FOSB. In SSC, the oncogenes ECT2 and DCUN1D1 were upregulated. In SCC, tumor suppressors DLG1 and DLGAP5 were upregulated, and TGFBR2 was downregulated. No significant changes were detected in DNA repair genes in ADC, in apoptotic genes in ADC or SCC, or in tumor suppressor genes in ADC. Pathway signatures of lung ADC and SCC show both similarities and differences to triple-negative breast cancer (TNBC). Significant changes in NAD metabolism appear to be unique to TNBC and ADC. Conclusions: Lung ADC and SCC are characterized by distinct alterations in DNA repair, cell cycle regulation, and metabolism, thus providing a molecular basis for novel therapeutic strategies.

Digital Transcript Profile Analysis with aRNA-LongSAGE Validates FERMT1 As a Potential Novel Prognostic Marker for Colon Cancer

To use gene transcript profiling to identify cancer-associated gene expression. Methods included (i) marker discovery using laser capture microdissection (LCM)-assisted specimen preparation and antisense RNA-long serial analysis of gene expression (aRNA-LongSAGE) on matched colon cancer and uninvolved colon tissue specimens (n = 5). Candidate tumor-associated genes were selected by combining the LongSAGE libraries reported herein with our previous colon cancer LCM-microarray transcript profiling data; (ii) marker selection and validation by quantitative real-time PCR (n = 15) and immunohistochemistry (n = 31); and (iii) independent validation on multiple tissue microarray (n = 203). Among 30 upregulated and 73 downregulated genes, upregulation of fermitin family member 1 (FERMT1), adenosylhomocysteinase (AHCY), secernin 1 (SCRN1), and SAC3 domain-containing protein 1 (SAC3D1) expression and downregulation of IgJ and MALL expression in colon cancer were confirmed by quantitative PCR. FERMT1 and AHCY protein expression was also upregulated in colon cancer compared with uninvolved colon mucosa, and FERMT1 expression showed upregulation in colon adenoma. Patients with moderate/strong tumor FERMT1 protein expression (n = 122) showed significantly poorer overall survival (OS; P = 0.011) and disease-free survival (DFS; P = 0.005) than patients with negative/weak tumor FERMT1 protein expression (n = 81). Multivariate Cox regression analysis showed that FERMT1 protein expression was also an independent prognostic factor for OS (P = 0.018) and DFS (P = 0.009). In addition, upregulated FERMT1 protein expression appeared to have some specificity among alimentary tract tumors. FERMT1 is a novel prognostic factor for colon carcinoma.

PUMA-mediated intestinal epithelial apoptosis contributes to ulcerative colitis in humans and mice

Intestinal epithelial cell (IEC) apoptosis contributes to the development of ulcerative colitis (UC), an inflammatory bowel disease (IBD) that affects the colon and rectum. Therapies that target the inflammatory cytokine TNF have been found to inhibit IEC apoptosis in patients with IBD, although the mechanism of IEC apoptosis remains unclear. We therefore investigated the role of p53-upregulated modulator of apoptosis (PUMA), a p53 target and proapoptotic BH3-only protein, in colitis and IEC apoptosis, using patient samples and mouse models of UC. In UC patient samples, PUMA expression was elevated in colitis tissues relative to that in uninvolved tissues, and the degree of elevation of PUMA expression correlated with the severity of colitis and the degree of apoptosis induction. In mice, PUMA was markedly induced in colonic epithelial cells following induction of colitis by either dextran sulfate sodium salt (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). The induction of PUMA was p53-independent but required NF-κB. Absence of PUMA, but neither absence of p53 nor that of another BH3-only protein (Bid), relieved DSS- and TNBS-induced colitis and inhibited IEC apoptosis. Furthermore, treating mice with infliximab (Remicade), a clinically used TNF-specific antibody, suppressed DSS- and TNBS-induced PUMA expression and colitis. These results indicate that PUMA induction contributes to the pathogenesis of colitis by promoting IEC apoptosis and suggest that PUMA inhibition may be an effective strategy to promote mucosal healing in patients with UC.

New Insights Into the Pathogenesis of Inflammatory Bowel Disease: Transcription Factors Analysis in Bioptic Tissues From Pediatric Patients

Our work is aimed at identifying ex vivo new transcription factors, potentially involved in the pathogenesis of pediatric inflammatory bowel disease (IBD), by using a microarray approach. Microarray, including 84 transcription factors, was performed in inflamed and uninflamed mucosal tissues of pediatric patients with Crohn disease (CD) and in healthy controls. Real-time polymerase chain reaction was used to confirm microarray results on a larger size of CD and patients with ulcerative colitis (UC). Protein expression was evaluated by Western blot assay. Microarray assay showed 40 genes differentially regulated in the inflamed mucosa and 17 in the uninflamed mucosa of patients with CD as compared with controls. Real-time polymerase chain reaction analysis revealed 10 transcripts in CD and 4 in UC, selected among those with higher differences as compared with healthy controls, significantly overexpressed in the inflamed tissues of patients. Moreover, 4 transcripts in CD and 2 in UC were found significantly upregulated in the uninvolved tissue. A further investigation evidenced an increased protein expression of activating transcription factor 3 and hypoxia-inducible transcription factor-1α in patients with CD as well as in Caco2 cell line stimulated by cytokines and hypoxia. The present study shows an evident upregulation of several transcription factors in the inflamed and uninflamed mucosa of children with IBD, suggesting that the inflammatory process is somehow activated at molecular levels even in the macroscopically normal mucosa of patients. A differential pattern of gene expression between CD and UC indicates distinct molecular mechanisms underlying the pathogenesis of 2 diseases. Finally, activating transcription factor 3 and hypoxia-inducible transcription factor-1α are proposed as new transcription factors potentially involved in the onset and maintenance of IBD.

The Nuclear-Factor κB Pathway Is Activated in Pterygium

Pterygium is a prevalent ocular surface disease with unknown pathogenesis. The authors investigated the role of nuclear factor kappa B (NF-κB) transcription factors in pterygium. Surgically excised primary pterygia were studied compared with uninvolved conjunctiva tissues. NF-κB activation was evaluated using Western blot analysis, ELISA, and DNA-binding assays. Primary pterygium fibroblasts were treated with TNF-α (20 ng/mL), and NF-κB activation was evaluated using immunocytochemistry, Western blot analysis, phospho-IκBα ELISA, and DNA-binding assays. TNF-α stimulation of NF-κB target genes RelB, NFKB2, RANTES, MCP-1, ENA-78, MMP-1, MMP-2, and MMP-3 in pterygium fibroblasts was compared with that in primary tenon fibroblasts by real-time PCR. Phosphorylation of IκBα (Ser32) was increased in pterygia tissues compared with uninvolved conjunctiva tissues, as determined by Western blot analysis and ELISA. IκBα expression was decreased, whereas nuclear RelA and p50 DNA-binding capacities were increased. Within 30 minutes of treatment with TNF-α, pterygium fibroblasts showed increased IκBα phosphorylation and nuclear translocation of RelA and p50. Treatment with TNF-α beyond 12 hours resulted in increased nuclear expression of RelB, p100, and p52. Furthermore, the upregulation of RANTES, MCP-1, ENA-78, MMP-1, MMP-2, and MMP-3 expression was more pronounced in TNF-α-treated pterygium fibroblasts than in tenon fibroblasts. The NF-κB pathway is shown for the first time to be activated in pterygia tissues compared with normal conjunctiva tissues. Stimulation by the inflammatory cytokine TNF-α can activate both canonical and noncanonical NF-κB pathways in pterygium fibroblasts with concomitant upregulation of NF-κB target genes.

Sexual dimorphism in LEC rat liver: Suppression of carbonic anhydrase III by copper accumulation during hepatocarcinogenesis

We examined age-related changes in the protein expression of carbonic anhydrase III (CAIII) in livers of Long-Evans with a cinnamon-like color (LEC) rats using an agouti color (LEA) rats as controls. The levels of the protein of CAIII in the liver of LEC male rats increased before 20 weeks of age, at the stage of acute hepatitis, and were decreased at 54 weeks of age, while those of CAIII in the liver of LEA male rats were highly expressed at all ages. In the normal LEA rats, CAIII showed sexual dimorphism. The level of CAIII in LEA male rat liver relative to female was four times higher. On the other hand, young LEC rat (at 4-12 weeks) showed a higher protein level of CAIII than LEA rats, and then decreased during development of hepatitis. CAIII mRNA also decreased in the LEC rat liver during hepatocarcinogenesis. The level of CAIII in the tumor region was lower than that in the tumor-free region. Immunohistochemical analysis showed that glutathione S-transferase P (GST-P) was positive and CAIII was negative in the precancerous region. The expression of CAIII was suppressed in cancerous lesions in hepatoma-bearing LEC rat liver compared to uninvolved surrounding tissues. These results indicated that suppression of CAIII accompanied hepatocarcinogenesis and it is a secondary consequence of the high copper levels in the liver.