Altered expression of CD44 and DKK1 in the progression of Barrett's esophagus to esophageal adenocarcinoma

Barrett's esophagus (BE) is an acquired condition in which the normal lining of the esophagus is replaced by intestinal metaplastic epithelium. BE can evolve to esophageal adenocarcinoma (EAC) through low-grade dysplasia (LGD) and high-grade dysplasia (HGD). The only generally accepted marker for increased risk of EAC is the presence of HGD, diagnosed on endoscopic biopsies. More specific markers for the prediction of EAC risk are needed. A tissue microarray was constructed comprising tissue samples from BE, LGD, HGD, and EAC. Marker expression was studied by immunohistochemistry using antibodies against CD44, DKK1, CDX2, COX2, SOX9, OCT1, E-cadherin, and β-catenin. Immunostaining was evaluated semi-quantitatively. CD44 expression decreased in HGD and EAC relative to BE and LGD. DKK1 expression increased in HGD and EAC relative to BE and LDG. CDX2 expression increased in HGD but decreased in EAC. COX2 expression decreased in EAC, and SOX9 expression increased only in the upper crypt epithelial cells in HGD. E-cadherin expression decreased in EAC. Nuclear β-catenin was not significantly different between BE, LGD, and HGD. Loss of CD44 and gain of DKK1 expression characterizes progression from BE and LGD to HGD and EAC, and their altered expression might indicate an increased risk for developing an EAC. This observation warrants inclusion of these immunohistochemically detectable markers in a study with a long patient follow-u

Serrated polyps of the colorectum: is sessile serrated adenoma distinguishable from hyperplastic polyp in a daily practice?

The distinction between serrated polyps of the colon is complex, particularly between hyperplastic polyps (HP) and sessile serrated adenomas (SSA). Recent data show that SSA might be the precursors of serrated colonic cancers, underlining the necessity of identifying them. We characterized the demographic and pathologic characteristics of 102 serrated lesions among 321 polyps of the colorectum and determined if SSA can be microscopically distinguished from HP in biopsy material of a daily practice. There were 81 HP (79%) and 7 SSA (7%) of which one displayed low-grade dysplasia. Only six serrated polyps (6%) could not be correctly classified. The main architectural criteria for distinguishing SSA from HP is the serrated feature along the crypt axis and the rarity of undifferentiated cells in the lower third of the crypts. SSA was significantly more often located in the right colon and larger (median, 11 vs 4 mm) than HP. SSA are rare serrated polyps that can be distinguished from HP based on their morphology, location in the right colon, and larger size. One SSA of our series showed low-grade dysplasia supporting the concept that this lesion might be a precursor of serrated adenocarcinom

Immunohistochemical localization of hTERT protein in human tissues

Telomerase is a ribonucleoprotein complex mainly composed of a reverse transcriptase catalytic subunit (telomerase reverse transcriptase gene, hTERT) that copies a template region of its RNA subunit to the end of the telomere. For detecting telomerase activity in a tissue specimen the TRAP assay is a relatively sensitive and specific method, but it can be used only on fresh tissue extracts and offers no information at the single cell level. Immunohistochemistry (IHC) allows to detect hTERT protein expression at an individual cell level in human tissues. We have tested commercially available anti-hTERT antibodies in formalin-fixed and paraffin-embedded human tissues by IHC. Only one monoclonal antibody (NCL-hTERT; Novacastra) was sufficiently specific and this was applied to human tissues in which telomerase activity had been shown by TRAP assay and hTERT mRNA expression by RT-PCR. hTERT protein localized diffusely in the nucleoplasm and more intensely in the nucleoli of cancer cells and proliferating normal cells. Mitotic cells showed diffuse staining of the entire cell. Granular cytoplasmic staining was occasionally found in some tumor cells. In telomerase-positive tumors not all the tumor cells showed hTERT immunoreactivity. A significantly heterogeneous hTERT protein expression was observed in human tumor tissues. The hTERT immunostaining in fixed tissues was concordant with telomerase activity and hTERT mRNA expression in corresponding non-fixed samples. Quantitative RT-PCR of microdissected sections showed that hTERT mRNA expression was higher in cells with nuclear expression than in those with cytoplasmic expression. Double staining with the M30 antibody showed that a subpopulation of hTERT-negative cells is apoptotic. We conclude that: (1) hTERT protein can be detected by IHC in fixed human tissues, but the choice of the antibody, tissue processing, and reaction conditions are critical, (2) hTERT protein localizes in the nucleoplasm, more strongly in the nucleolus, and occasionally in the cytoplasm, (3) telomerase-positive tumors show significant heterogeneity of hTERT protein expression, and (4) a subpopulation of hTERT protein negative tumor cells is identified as apoptotic cell

Stripping of X ray bremsstrahlung spectra up to 300 kV_p on a desk type computer.

The direct result of a spectrometric measurement is a pulse height distribution. In the energy region up to 300 keV three corrections in particular need to be applied to get the photon spectrum: corrections for K-escape, Compton scattering and inefficient photon absorption. A simple 'stripping' procedures is described which may be implemented on a desk type computer. All data necessary are either available in the literature or may be derived from the measurement of very heavily filtered X-ray spectra. The accuracy of the procedure is better than ± 5% of the peak value. Results are compared with a more detailed stripping procedure, based on Monte Carlo calculated data

An agarose matrix facilitates sectioning of tissue microarray blocks.

Tissue microarray (TMA) is a powerful, high-throughput technique for in situ investigation of biomarkers in many tissue samples in a paraffin block by immunohistochemistry or fluorescence in situ hybridization (FISH), and has rapidly become the standard in marker studies. One of the difficult steps in the procedure is the sectioning of array blocks and mounting of sections using special slides and/or adhesive-coated tape, which demands specific experience and is time-consuming. We report an arraying method that allows melting of the receiving paraffin block and subsequent sectioning like any ordinary paraffin-embedded tissue block. The major difference from the standard microarray technique is the use of an agarose matrix in the recipient block. The agarose matrix allows melting of the paraffin without disturbing the array, resulting in perfect integration of the tissue cores. The agarose-paraffin TMA blocks limit tissue core loss during cutting, mounting, or immunohistochemical or FISH staining and better maintains the array

MLC positioning checks and calibration with a portal imaging system

For checking the leaf positions of a MLC (Multi Leaf Collimator) images are acquired with an EPID (Electronic Portal Imaging Device) and then evaluated with a programme (MLC check) developed in-house. During image acquisition a Perspex tray with two metal markers of known position (in the radiation field) is inserted in the satellite tray holder. After determination of the marker positions within the image coordinate system, the image can be transformed to the radiation field coordinate system. This allows the exact determination of the leaf tip positions relative to the radiation field. This evaluation can be applied to images of arbitrary field shapes, provided they were acquired in the same geometry (EPID position, gantry and collimator angles). The entire measurement procedure is based on images in bmp (Windows Bitmap) format, with a 1024 x 1024 matrix and a pixel depth of 24 bit (8 bit per color channel). A suitable marker plate can be manufactured without sophisticated workload; thus the method may be easily and cost effectively adapted at other locations

Aberrant crypt foci in patients with neoplastic and nonneoplastic colonic disease.

Aberrant crypt foci (ACF) are putative preneoplastic lesions that might represent the earliest morphological lesion visible in colonic carcinogenesis. However, findings concerning the growth and morphological features of these lesions in human studies suggest that ACF are highly heterogeneous in nature. In this study, we evaluated the morphological features of a large number of ACF in colon mucosa of 26 patients with colorectal carcinoma (CRC), four patients with adenoma as well as seven patients with nonneoplastic colonic diseases. By dissecting microscope, 508 ACF were identified, and of these, 378 were sampled for histological examination. The median ACF density (number of ACF/cm2) was significantly higher in the left colon than in the right colon (0.047 v 0.014 ACF/cm2). Unexpectedly, in our series, the overall ACF density was higher in the nonneoplastic colonic diseases than in CRC (0.13 v 0.032 ACF/cm2, P=.0087), cases of nonneoplastic diseases, however, being limited to 7 patients. ACF were significantly larger in colons with CRC or adenoma than in colons with nonneoplastic disease (P < .03). On histological examination, we observed 133 ACF with normal epithelium, 189 ACF with hyperplasia, 27 ACF with atypical hyperplasia, and 29 ACF with dysplasia. We noted a progressive increase of median ACF size from normal mucosa to hyperplasia, atypical hyperplasia, and dysplasia. Dysplastic ACF were more frequently observed in patients with CRC or adenoma and showed predominantly elongated crypt orifices (P < .0001). We conclude that ACF are histologically heterogeneous, encompass a spectrum of lesions of which only a subset are associated with dysplasia and then represent an early step in colorectal carcinogenesis. ACF with dysplasia are characterized by larger size, elongated crypt orifices, and an association with CRC