This retrospective study indicates that Ki-67 and ploidy may have stronger prognostic impact on OS and DFS than other parameters investigated after adjusting for stage and tumor grade. Prospective studies to elucidate the mechanism and prognostic significance of these findings are necessary.
Apoptosis or programmed cell death is often altered in malignancies and is frequently determined by the terminal transferase-mediated nick end labeling technique (TUNEL). However, commercially available protocols can produce high background and false-positive staining, which renders the distinction between apoptosis and necrosis difficult. In an attempt to develop a rapid and reproducible method for detecting and quantifying apoptosis, we coupled optimization of the Apoptag Plus Peroxidase In Situ Apoptosis Detection kit with quantitative histomorphometric computer imaging software using the Bacus Laboratories Incorporated Slide Scanner (BLISS). Multiple (200 -350) unique 40؋ images were scanned using the BLISS system and downloaded into the WebSlide Browser program, creating a permanent, scanned record of the area assessed. The stored images were counted, with the final analysis simultaneously taking into account cells that were immunohistochemically positive and the histology of the surrounding cells to reduce the possibility of false positive and negative staining. In addition, cells with equivocal staining can be simultaneously reviewed by other technologists with networked WebSlide Browser access to the same images. Our data show that the advantages offered by the BLISS imaging software greatly reduce the potential drawbacks of using the TUNEL method as a sole means of quantification.
Aims-Several gastrointestinal motility diseases are associated with altered numbers of interstitial cells of Cajal (ICC) and testing for alterations in numbers of ICC has been proposed as one way to improve routine diagnostics in motility diseases. However, the protocols currently used to visualize ICC in formalin fixed, paraffin embedded (FFPE) tissue using antibodies to CD117 have not been optimized for studying motility disorders. Therefore, the aims of this study were to determine the optimal protocol using FFPE tissue, determine normal values for ICC in non-neoplastic human colon, and compare results to those obtained using immunofluorescence (IF).Methods/Results-Non-neoplastic tissue was collected from patients undergoing resection for colon cancer and fixed for both light (FFPE) and IF testing. Sections were processed for standard immunohistochemistry using different primary antibodies in conjunction with variations in antigen retrieval (EDTA, citrate), antibody dilution, blocking and detection (Mach2, Mach3, Envision+). Best results were obtained with EDTA retrieval, DAKO CD117 antibody and Mach3 detection. Conclusions-The optimized protocol presented improved CD117 detection in FFPE tissues and showed good concordance with overall localization of CD117-immunoreactive (IR) ICC as detected by IF. As such, this protocol may be more useful than current procedures for diagnostics in motility disorders.
10022 Background: Patients with colon cancers displaying high frequency microsatellite instability (MSI-H) are reported to have a favorable prognosis compared to microsatellite stable (MSS) tumors. However, prognostic factors underlying this observation are poorly understood. We studied apoptotic and proliferative indices and their relationship to MSI status, clinicopathological features, and patient survival rates. Methods: Archival Dukes’ stage B2 (n=83) and C (n=246) primary colon adenocarcinomas from patients enrolled in five 5-fluorouracil-based adjuvant therapy trials were analyzed for MSI using a PCR-based assay (MSI-H: ≥30% of loci with instability), and expression of hMLH1, hMSH2 and p53 proteins by immunostaining. Apoptosis was analyzed by the TUNEL assay and the proliferative index (PI: S phase + G2M) and DNA ploidy by flow cytometry. Correlations between markers and associations with overall survival (OS) censored at 8 yrs were sought. Results: MSI-H (n=58.18%) tumors were more likely proximal, diploid, high grade, p53 negative (vs. MSS/MSI-L; all p<0.0001), and from women (p=0.002). Of 58 MSH-H cases tested, 54 showed loss of hMLH1 (n=50) or hMSH2 (n=4) proteins. Median proliferative index (PI) [12.6 vs. 17.4; p=0.0002] was reduced in MSI-H versus MSS/MSI-L tumors. Lower PI was associated with diploidy (p<0.0001) and negative p53 expression (p=0.003). Apoptotic indices (AIs) were increased in MSI-H cancers (vs. MSS/MSI-L, p=0.082), as was the AI/PI ratio (p=0.0065). Interestingly, AI (p=0.02) and AI/PI (p<0.0001) were significantly increased in diploid versus nondiploid tumors, and after removal of MSI-H cases, relationships held for PI and AI/PI with ploidy. Better OS was related to MSI-H (p=0.032), loss of hMLH1 or hMSH2 (p=0.024), diploidy (p=0.0015), and lower PI (p=0.078), but not AI, AI/PI, nor p53 (adjusting for stage, grade, treatment and stratifying by study). Conclusions: MSI-H tumors are characterized by reduced proliferative indices and a higher ratio of apoptosis to proliferation, reflecting slower tumor growth rates compared to MSS/MSI-L tumors. These features may contribute to their better survival. Lower PI and increased AI/PI are also features of diploid MSS cancers. [Table: see text]
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