Exosomal Protease Cargo as Prognostic Biomarker in Colorectal Cancer.

OBJECTIVE: The aim of the study was to develop a model for predicting cancer risk in colorectal polyps' patients (CPPs), as well as to reveal additional prognosis factors for Stage III colorectal cancer based on differences in subpopulations of tetraspanins, tetraspanin-associated and tetraspanin-non-associated proteases in blood plasma exosomes of CPPs and colorectal cancer patients (CRCPs). METHODS: The subpopulations of CD151- and Tspan8-positive exosomes, the subpopulations of metalloproteinase at the surface of СD9-positive exosomes and the level of 20S proteasomes in plasma exosomes in 15 CPPs (tubulovillous adenomas) and 60 CRCPs were evaluated using flow cytometry and Western blotting. Logistic regression analysis was performed to predict cancer risk of CPPs. RESULTS: The levels of 20S proteasomes in exosomes, MMP9+, MMP9+/MMP2+/EMMPRIN+ in CD9-positive blood plasma exosomes are associated with the risk of malignant transformation of colorectal tubulovillous adenomas.  In patients with Stage III CRC, the levels of 20S proteasomes (less than 2 units) and MMP9+ subpopulations (more than 61%) in plasma exosomes are unfavorable prognostic factors for overall survival. The levels of 20S proteasomes and ADAM10+/ADAM17- subpopulations in CD9-positive blood plasma exosomes are the most significant values for predicting relapse-free survival. CONCLUSION: Protease cargo in CD9-positive blood plasma exosomes is prognostic biomarker for colorectal polyps and colorectal cancer.

Incidental focal uptake in colorectal location on oncologic ¹8FDG PET and PET/CT studies: histopathological findings and clinical significances.

PURPOSE: Unexpected focal colonic or rectal radiotracer activity is an usual finding in patients subjected to a PET study. The aim of this work has been to evaluate the clinical significance of this finding in the prediction of an existing colorectal malignancy. MATERIAL AND METHODS: During the last three years, all patients studied with (18)F-FDG PET/CT and PET for oncologic work-up purposes were prospectively surveyed for focal colorectal radiotracer activity. Colonoscopy was performed in all patients with this incidental finding in order to exclude colonic malignancy. CEA level, maximum standardized uptake value (SUVmax), CT findings, colonoscopy findings and histopathological results were prospectively analyzed in all patients. RESULTS: A total of 2290 patients were evaluated, 158 of whom were studied with PET and the remainder with a hybrid PET/CT. Focal FDG colorectal activity was incidentally detected in 27 patients with no previous history of colorectal cancer. Colorectal adenocarcinoma was diagnosed in seven (25.9%) patients. A pre-cancerous lesion was found in eleven patients (40.7%). Eight patients (29.6%) had no macroscopic lesions. One patient was diagnosed with a benign lesion. Any focal activity found in the colon by (18)F-FDG PET/CT examination predicts a probability greater than 50% of an underlying malignant or premalignant lesion in the histopathological analysis (logistic regression, p=0.01), independently of the calculated SUVmax. CONCLUSION: According to the results of the present study, we recommend the performance of a colonoscopy and biopsy of any suspicious lesions, in all patients with unexpected focal FDG activity found in colon or rectum during a (18)F-FDG PET/CT examination.

[Villous adenoma of the urinary tract: a clinicopathological study].

OBJECTIVE: To explore the clinicopathological features, immunophenotype, differential diagnosis, pathogenesis and prognosis of villous adenoma with poorly differentiated adenocarcinoma of the urinary tract. METHODS: Clinical and pathologic findings of 3 cases of villous adenoma with poorly differentiated adenocarcinoma of the urinary tract were analyzed by gross examination, microscopic investigation and immunohistochemical staining. The related literatures were reviewed. RESULTS: All of the three cases were middle-aged or elderly patients. Three cases all presented with hematuria and mucusuria. Endoscopic examination identified that case 1 had a polyp with broad attachment in the dome of bladder, case 2 had a solid mass in the ureter, and case 3 had a exophytic fungating tumor in the renal pelvis. Microscopically, case 1 revealed a papillary lesion with finger-like processes lined by pseudostratified columnar epithelium with abundant goblet cells. The cells demonstrated moderate degree dysplasia. In case 2 and case 3, both villous adenomas and poorly differentiated adenocarcinoma were observed, the adenoma cells arranged in a cribriform pattern, and the tumor cells showed severe atypia, mitotic activity, and transition with invasive poorly differentiated adenocarcinoma. Immunohistochemically, the tumor cells in three cases were positive for CK20, CEA,EMA and MUC-1; none of them expressed cdx-2 and PSA; In case 2 and 3, the same immunophenotype of villous adenomas and their associated adenocarcinomas was observed, but the number of the positive cells of p53 and Ki-67 staining were significantly increased in the area of adenocarcinomas than in that of the villous adenomas. CONCLUSIONS: Villous adenoma of the urinary tract is rare. It can occur in the urinary bladder, urachus, renal pelvis, ureter and urethra. These lesions may have malignant potential and frequently coexist with other malignant tumors. So, villous adenoma of the urinary tract should be removed completely and sampled thoroughly to avoid missing a more aggressive component.

The McKittrick-Wheelock syndrome: a rare cause of chronic diarrhoea.

The McKittrick-Wheelock syndrome, a rare disorder, is caused by fluid and electrolyte hypersecretion from a rectal tumour and patients can develop a depletion syndrome characterised by severe dehydration, hyponatraemia, hypokalaemia and metabolic acidosis. We present a case of a 62-year-old man who presented with chronic diarrhoea that had resulted in numerous previous hospital admissions. On physical exam, the patient showed signs of volume depletion. A soft polypoid mass was appreciated on digital rectal examination. Laboratory tests showed renal failure and significant electrolyte abnormalities. Colonoscopy revealed a large, friable mass in the rectosigmoid region. Biopsies were consistent with tubolovillous adenoma. Subsequently, the patient underwent surgical resection, which on pathology exhibited evidence of high-grade dysplasia, and the patient was diagnosed with McKittrick-Wheelock syndrome. It is essential to identify this condition in a timely manner as it is associated with high morbidity and complications, some of which may be life threatening.

Efficient recovery of proteins from multiple source samples after TRIzol(®) or TRIzol(®)LS RNA extraction and long-term storage.

BACKGROUND: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol(®) and TRIzol(®)LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation. RESULTS: TRIzol(®)LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol(®) was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer's disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol(®)-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at -80°C until used for protein isolation.Simple modifications to the TRIzol(®) manufacturer's protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol(®) manufacturer's protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol(®)-chloroform fractions stored for up to 2 years at -80°C. CONCLUSIONS: We provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol(®) and TRIzol(®)LS compared to the manufacturer`s protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer's disease mice model, stored in TRIzol(®)-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol(®) manufacturer`s protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

Vaginal tubulovillous adenoma: a clinicopathologic and molecular study with review of the literature.

Vaginal villous or tubulovillous adenomas (TVA) are uncommon tumors histologically similar to their intestinal counterparts. After reviewing the literature, we report the eighth case of TVA, which presented as a polypoid tumor in the vagina, at suburethral level, in a 19-yr-old woman with Arnold-Chiari type II malformation and a myelomeningocele at birth. The tumor consisted of long villi lined by columnar cells with brush borders, pseudostratified nuclei, and foci of high-grade atypia. Immunohistochemistry was positive for cytokeratin 7, estrogen and progesterone receptors, CA19.9, p16, p53, and Ki-67 (53%), with a normal membranous pattern for ß-catenin, but negative for cytokeratin 20, CDX2, carcinoembryonic antigen, chromogranin A, and synaptophysin. Neither human papillomavirus nor mutations in the K-RAS, BRAF, or LKB1/STK11 genes were detected. Although a rare neoplasm, awareness of this tumor is important as it must be distinguished from colonic adenocarcinoma or other malignant or benign conditions. The existence of 2 previously reported malignant cases merging with TVAs, and the presence of foci of high-grade dysplasia (p53-positive) in the present case, support TVA as a premalignant lesion.

Multiple tubulovillous adenomas of the vulva.

Enteric-type lesions are rare in the female genital tract. We report the first case of multiple vulvar tubulovillous adenomas with transformation into adenocarcinoma. A 31-year-old woman presented with recurrent vulvar polypoid lesions resembling condylomas that were excised. These tumors were characterized by their tubulovillous architecture and intestinal differentiation, with columnar epithelium, goblet cells, and Paneth cells. As in their colonic counterpart, the degree of dysplasia was evaluated. The lesions consisted of 3 low-grade adenomas and 1 adenocarcinoma with superficial invasion. After 15 months, there is no sign of recurrence. The clinical presentation, pathological findings, differential diagnoses, and pathogenesis are discussed.

A case of villous adenoma of the urinary bladder with tubulovillous architecture: characterization by immunohistochemical analysis.

Villous adenomas of the urinary tract are rare as compared to urothelial carcinoma. We report a case of urinary bladder villous adenoma in a 90-year-old woman. Cystoscopic examination revealed a papillary tumour in the diverticulum at the posterior wall of the urinary bladder. Transurethral resection was performed and histopathological examination revealed predominantly tubulovillous architecture, and showed an identical immunohistochemical profile to villous adenoma associated with cystitis glandularis.

Tubular adenomas with minor villous changes show molecular features characteristic of tubulovillous adenomas.

Advanced colorectal polyps are identified based on size ≥10 mm, high-grade dysplasia, and/or villous histology. A diagnosis of tubular adenoma (TA) is recommended if villous change occupies <20% of the lesion, or tubulovillous adenoma (TVA) is recommended if there is 20% to 80% villosity. We hypothesized that even subtle villous changes (1% to 20%) would correlate with advanced molecular features. Two hundred sixty-nine colorectal adenomas were examined for KRAS and BRAF mutation and immunohistochemical staining of ß-catenin, O6-Methyl Guanine DNA Methyltransferase (MGMT), and p53. Adenomas were classified as TA1 (0% villosity, n=70), TA2 (1% to 20% villosity, n=81), or TVA (21% to 80% villosity, n=118). Clinical and molecular features were analyzed by univariate χ² and multivariate logistic regression. There was an incremental increase in KRAS mutation frequency with increasing villous compartment (17.9% TA1, 59.0% TA2, 78.4% TVA; P<0.001). MGMT was more frequently lost in TA2 (37.0%) than in TA1 (8.6%) (P<0.001) but did not differ from TVA (39.8%). p53 overexpression was more common in TA2 (38.3%) than in TA1 (10.0%) (P<0.001) but did not differ from TVA (32.2%). On multivariate analysis, TA2 adenomas were more likely to have a KRAS mutation [odds ratio (OR) 6.6, 95% confidence interval (CI), 3.0-14.2], MGMT loss (OR 6.2, 95% CI, 2.4-16.0), or p53 overexpression (OR 5.6, 95% CI, 2.3-13.7) than TA1. We have identified a subgroup of TAs based on subtle villous changes. These adenomas are more likely to show molecular features that are characteristic of TVAs than TAs. These data support the concept that any villous change may indicate increased malignant potential and may be useful to consider when assigning surveillance guidelines.

[Serrated lesions of colon and their malignant potential].

OBJECTIVE: To study the serrated lesions of colon and to compare the malignant potential between traditional serrated adenomas (TSA) and conventional adenomas (CAD). METHODS: A total of 5347 cases of colorectal polyps encountered in five regional hospitals during a five-year period were retrospectively reviewed. The serrated lesions were classified on the basis of histologic examination. One hundred and eighty-seven cases of CAD (including 160 cases of tubular adenoma and 27 cases of villous adenoma) and 36 cases of invasive adenocarcinoma were randomly selected as the controls. The degree of dysplasia and expressions of Ki-67, p53 and beta-catenin in TSA and CAD were compared. RESULTS: Amongst the 5347 colorectal polyps studied, 258 cases (4.8%) of serrated lesions were found, which included 112 cases (43.4%, 112/258) of hyperplastic polyp, 78 cases (30.2%, 78/258) of TSA and 26 cases (10.1%, 26/258) of sessile serrated adenoma. Sixty-two cases of TSA were identified from 3 hospitals, in which moderate dysplasia was found in 13 cases. High-grade intraepithelial neoplasia and ICA were found in 6 cases (9.6%). Compared with the 187 cases of CAD, moderate dysplasia were found in 27 cases and high-grade intraepithelial neoplasia and invasive adenocarcinoma were found in 25 cases (13.3%, χ(2) = 19.373, P = 0.000). There was statistically significant difference between TSA and CAD in the degree of dysphasia. The expression of Ki-67, p53 and beta-catenin in TSA and CAD showed no significant difference (P > 0.05). CONCLUSIONS: The incidence of serrated lesions is lower in northern Chinese population than that in Caucasians. TSA has obvious malignant potential; but the rate associated with high-grade intraepithelial neoplasia and invasive adenocarcinoma is lower than that in CAD.

Differentiation of tubular and villous adenomas based on Wnt pathway-related gene expression profiles.

This study was undertaken to define whether differences in the expression of Wnt pathway components are present between normal colonic mucosa, early (tubular) adenomas and villous adenomas which have a higher malignant potential. Normal mucosa, tubular adenomas and villous adenomas were obtained from twelve patients. RNA was isolated and utilized for Wnt pathway-specific membrane array expression analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescent immunohistochemistry (IHC) were utilized for confirmatory analyses. Fifteen Wnt pathway-related genes showed differential expression between villous adenomas and normal mucosa and villous and tubular adenomas at a significance level of p<0.01. Genes involved in canonical Wnt (beta-catenin) signaling with increased expression in villous adenomas included wnt1, fz2, csnk2A2, pygo2, pygo1, frat2 and myc, the latter confirmed by qRT-PCR and IHC. Myc protein expression was confined primarily to stromal components of villous adenomas. Genes involved in non-canonical Wnt signaling with increased expression in villous adenomas included rho-u, daam1, damm2, cxxc4 and nlk. Successive increases in the expression of ctnnb1 (beta-catenin) from normal to tubular adenomas to villous adenomas was seen. The Wnt pathway gene expression profile can differentiate between tubular and villous adenomas. These data suggest that Wnt signaling regulation changes during the progression from normal mucosa to tubular adenomas to villous adenomas. Expression of Myc in adenoma stroma suggests a dynamic signaling network within adenomas between mucosal and stromal elements. Inhibition of the Wnt pathway may provide a novel approach for cancer prevention in patients with benign tubular adenomas.

[Clinicopathological and immunohistochemical features of colorectal sessile serrated adenoma].

OBJECTIVE: To investigate the clinicopathological characteristics and expression status of Ki67, p53, CEA, CDX, CK7 in colorectal sessile serrated adenoma (SSA). METHODS: The clinicopathological data of 11 cases of SSA, 51 cases of hyperplastic polyp (HP) and one case with mixed HP/SSA were reviewed and analyzed retrospectively. The expression of Ki67, p53, CEA, CDX and CK7 were detected by immunohistochemistry. RESULTS: The major histological features in SSA were architectural abnormality in crypts, dilatation of serrated crypt bases like an inverted "T" or "L" shape adjacent to muscularis mucosa. Atypical cells containing round to oval nuclei and nucleoli were also observed. The immunohistochemical staining showed that the expression of p53 increased gradually from HP to TA: 11.8% in HP, 20.0% in SSA, 41.2% in VTA and 75.0% in TA, with a significant difference among the groups (chi(2) = 17.996, P = 0.000). However, no significant difference in the expression of CDX and CK7 was observed between HP and SSA. Of the 10 SSA cases, positive expression of Ki67 was found in cells located in the base or middle part of crypt in 6 cases, positive cells index was 26% - 50% in 5 cases, and > 50% in 3. Compared with the expression of Ki67 in the HP, VTA and VA, SSA showed a significant difference in both the positive cell number and in the positive regions. (positive number: chi(2) = 34.601, P = 0.000; positive regions: chi(2) = 63.077, P = 0.000). CONCLUSION: Morphological diagnosis of SSA was mainly based on crypt architectural and cellular abnormalities, and the crypt architectural abnormality may be more important than cellular features. Detection of p53 and Ki67 expression may be helpful in differential diagnosis and understanding the nature of SSA.

[Clinicopathologic features and proliferative status of colorectal serrated lesions: a study of 104 cases].

OBJECTIVE: To study the clinicopathologic features and proliferative status of colorectal hyperplastic polyp (HP), sessile serrated adenoma (SSA) and traditional serrated adenoma (TSA). METHODS: One hundred and four cases colorectal serrated lesions were collected from 2628 cases of colorectal polyps during the period from November, 2002 to December, 2007. The clinicopathologic features and expression of proliferation marker Ki-67 were studied. RESULTS: On the basis of morphologic examination, 60 cases were classified as HP, 20 cases as TSA, 11 cases as SSA, 7 cases as mixed HP/SSA/TSA, and 6 cases as mixed serrated polyp/adenoma and tubular adenoma. Immunohistochemical study for Ki-67 showed that 40 cases (78%) of the 51 cases of HP were either mostly negative or rarely (<25% cells) positive. Most of the positive cells were located at crypt bases. Among the 15 cases of TSA, 11 of them revealed positive cryptal cells (25% to 50% or>50% positivity). Most of the positive cells were located in mid portion of crypts. The number and distribution of Ki-67 positive cells in SSA were similar to those in TSA but were significantly different from those in tubular adenoma and adenocarcinoma (chi2=34.601, P=0.000; chi2=63.077, P=0.000, respectively). CONCLUSIONS: HP, SSA and TSA have their morphologic characteristics, with some overlapping features noted. The distinction between SSA and HP can be difficult. Diagnosis of SSA relies mostly on architectural rather than cytologic features. The distinction between TSA and SSA depends mainly on the presence of dysplasia. Ectopic crypt formation is almost exclusively seen in TSA. The distribution and percentage of Ki-67-positive cells are also helpful in subtyping of various colorectal serrated lesions. In general, the proliferative index is lower in serrated adenoma (TSA or SSA) than in tubular adenoma.

Successful antiangiogenic combination therapy for pseudomyxoma peritonei with bevacizumab and capecitabine.

Effective systemic therapy for advanced pseudomyxoma peritonei (PMP) is the focus of investigation. We describe a case of PMP arising from an adenoma of the appendix in a 58-year-old man. First, the patient underwent explorative laparotomy with ileocoecal resection, but without possibility of major tumor debulking due to adhesive gross tumor masses. Subsequently, six cycles of Folfox IV chemotherapy were administered, without response, but with severe side effects. Upon progressive disease, a combination of bevacizumab and capecitabine led to a long term stabilization of disease and obvious improvement of performance status. Our case suggests that modulation of tumor microenvironment and angiogenesis by bevacizumab, potentially augmented by moochemotherapy, may be beneficial in borderline tumors such as PMP.

Deletion hotspots in AMACR promoter CpG island are cis-regulatory elements controlling the gene expression in the colon.

Alpha-methylacyl-coenzyme A racemase (AMACR) regulates peroxisomal beta-oxidation of phytol-derived, branched-chain fatty acids from red meat and dairy products -- suspected risk factors for colon carcinoma (CCa). AMACR was first found overexpressed in prostate cancer but not in benign glands and is now an established diagnostic marker for prostate cancer. Aberrant expression of AMACR was recently reported in Cca; however, little is known about how this gene is abnormally activated in cancer. By using a panel of immunostained-laser-capture-microdissected clinical samples comprising the entire colon adenoma-carcinoma sequence, we show that deregulation of AMACR during colon carcinogenesis involves two nonrandom events, resulting in the mutually exclusive existence of double-deletion at CG3 and CG10 and deletion of CG12-16 in a newly identified CpG island within the core promoter of AMACR. The double-deletion at CG3 and CG10 was found to be a somatic lesion. It existed in histologically normal colonic glands and tubular adenomas with low AMACR expression and was absent in villous adenomas and all CCas expressing variable levels of AMACR. In contrast, deletion of CG12-16 was shown to be a constitutional allele with a frequency of 43% in a general population. Its prevalence reached 89% in moderately differentiated CCas strongly expressing AMACR but only existed at 14% in poorly differentiated CCas expressing little or no AMACR. The DNA sequences housing these deletions were found to be putative cis-regulatory elements for Sp1 at CG3 and CG10, and ZNF202 at CG12-16. Chromatin immunoprecipitation, siRNA knockdown, gel shift assay, ectopic expression, and promoter analyses supported the regulation by Sp1 and ZNF202 of AMACR gene expression in an opposite manner. Our findings identified key in vivo events and novel transcription factors responsible for AMACR regulation in CCas and suggested these AMACR deletions may have diagnostic/prognostic value for colon carcinogenesis.