Analytical Cellular Pathology - Volume 31, issue 1 - Journals

HER-2/neu amplification testing in breast cancer by multiplex ligation-dependent probe amplification in comparison with immunohistochemistry and in situ hybridization

Article Type: Research Article

Abstract: Background: Assessment of HER-2/neu status in invasive breast cancer is crucial to establish eligibility for trastuzumab and taxane based chemotherapy. Next to immunohistochemistry (IHC) to evaluate protein overexpression, a second line gene amplification test is required for cases with equivocal protein expression. This study aimed to validate a new PCR based test, called Multiplex Ligation-dependent Probe Amplification (MLPA), as a simple and quick method to assess HER-2/neu gene amplification status in invasive breast cancer. Methods: MPLA results were compared with gene amplification status assessed by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as gold standard, …and with protein overexpression by IHC in 518 breast carcinoma patients. Results: About 10% of cases overexpressed HER-2/neu at the protein level (IHC), and 11% of cases showed gene-amplification by MLPA. A high concordance was found between FISH and CISH, MLPA and IHC, and MLPA and CISH. MLPA showed amplification in 7/36 (19%) of the equivocal IHC 2+ cases. However, of the IHC 0/1+ cases, 6/434 (1.4%) were also amplified by MLPA, and amplification was confirmed in all of these cases by FISH/CISH. On the other hand, one of the 48 (2%) IHC 3+ cases was normal by MLPA and lack of amplification was confirmed by FISH/CISH. Conclusion: MLPA is a fast, accurate and cheap method to detect breast cancer HER-2/neu amplification in small quantities of DNA extracted from paraffin blocks, and thereby a reliable alternative to FISH and CISH. Show more

Keywords: HER-2/neu, amplification, in situ hybridization, multiplex ligation dependent probe amplification, immunohistochem- istry

DOI: 10.3233/CLO-2009-0461

Citation: Analytical Cellular Pathology, vol. 31, no. 1, pp. 1-10, 2009

Price: EUR 27.50

Quantitative fluorescence determination of long-fragment DNA in stool as a marker for the early detection of colorectal cancer

Article Type: Research Article

Abstract: Background: A variety of molecular markers have been evaluated for the development of a non-invasive approach to the diagnosis of colorectal cancer. We aimed to validate the diagnostic accuracy, using the same threshold as in the previous pilot study, of fluorescent long DNA test as a relatively simple and inexpensive tool for colorectal cancer detection. Methods: A case-control study was conducted on 100 healthy subjects and 100 patients at first diagnosis of colorectal cancer. Human long-fragment DNA in stool was quantified by fluorescence primers and a standard curve and expressed in DNA nanograms. Results: We validated the 25-ng …value, which emerged as the most accurate cut-off in the pilot study, obtaining 79% (95% CI, 71–87%) sensitivity and 89% (95% CI, 83–95%) specificity. Specificity was very high for all cut-off values (15–40 ng) analyzed, ranging from 78 to 96%. Sensitivity was only slightly lower, reaching 84% at the lowest cut-off and maintaining a good level at the higher values. Diagnostic potential was independent of gender, age and tumor site. Conclusion: Fecal DNA analysis is a non-invasive and fairly simple test showing high diagnostic potential. These characteristics, together with the small amount of stool required, make it potentially suitable to be used alongside or as an alternative to current non-invasive screening approaches. Our next step will be to validate these results in a large-scale cohort study of a screening population, which is needed prior to implementation into clinical practice. Show more

Keywords: Fluorescence long DNA, stool, colorectal cancer, diagnosis

DOI: 10.3233/CLO-2009-0443

Citation: Analytical Cellular Pathology, vol. 31, no. 1, pp. 11-17, 2009

Price: EUR 27.50

Potential biomarkers of colorectal adenoma–dysplasia–carcinoma progression: mRNA expression profiling and in situ protein detection on TMAs reveal 15 sequentially upregulated and 2 downregulated genes…

Article Type: Research Article

Abstract: Background: As most colorectal cancers (CRC) develop from villous adenomas, studying alterations in gene expression profiles across the colorectal adenoma–dysplasia–carcinoma sequence may yield potential biomarkers of disease progression. Methods: Total RNA was extracted, amplified, and biotinylated from colonic biopsies of 15 patients with CRC, 15 with villous adenoma and 8 normal controls. Gene expression profiles were evaluated using HGU133Plus2.0 microarrays and disease progression associated data were validated with RT-PCR. The potential biomarkers were also tested at the protein level using tissue microarray samples of 103 independent and 16 overlapping patients. Results: 17 genes were validated to show sequentially …altered expression at mRNA level through the normal–adenoma–dysplasia–carcinoma progression. Prostaglandin-D2 receptor (PTGDR) and amnionless homolog (AMN) genes revealed gradually decreasing expression while the rest of 15 genes including osteonectin, osteopontin, collagen IV–alpha 1, biglycan, matrix GLAprotein, and von Willebrand factor demonstrated progressively increasing expression. Similar trends of expression were confirmed at protein level for PTGDR, AMN, osteopontin and osteonectin. Conclusions: Downregulated AMN and PTGDR and upregulated osteopontin and osteonectin were found as potential biomarkers of colorectal carcinogenesis and disease progression to be utilized for prospective biopsy screening both at mRNA and protein levels. Gene alterations identified here may also add to our understanding of CRC progression. Show more

Keywords: Colorectal cancer, adenoma–dysplasia–carcinoma sequence, gene expression signature, whole genomic oligonucleo- tide microarray, tissue microarray, amnionless homolog, prostaglandin D2 receptor

DOI: 10.3233/CLO-2009-0458

Citation: Analytical Cellular Pathology, vol. 31, no. 1, pp. 19-29, 2009

Price: EUR 27.50

Genomic profiling by array comparative genomic hybridization reveals novel DNA copy number changes in breast phyllodes tumours

Article Type: Research Article

Abstract: Breast phyllodes tumour (PT) is a rare fibroepithelial tumour. The genetic alterations contributing to its tumorigenesis are largely unknown. To identify genomic regions involved in pathogenesis and progression of PTs we obtained genome-wide copy number profiles by array comparative genomic hybridization (CGH). DNA was isolated from fresh-frozen tissue samples. 11 PTs and 3 fibroadenomas, a frequently occurring fibroepithelial breast tumour, were analyzed. Arrays composed of 2464 genomic clones were used, providing a resolution of ~1.4 Mb across the genome. Each clone contains at least one STS for linkage to the human genome sequence. No copy number changes were …detected in fibroadenomas. On the other hand, 10 of 11 PT (91%) showed DNA copy number alterations. The mean number of chromosomal events in PT was 5.5 (range 0–16) per case. A mean of 2.0 gains (range 0–10) and 3.0 losses (range 0–9) was seen per case of PT. Three cases showed amplifications. DNA copy number change was not related to PT grade. We observed recurrent loss on chromosome 1q, 4p, 10, 13q, 15q, 16, 17p, 19 and X. Recurrent copy number gain was seen on 1q, 2p, 3q, 7p, 8q, 16q, 20. In this study we used array CGH for genomic profiling of fibroepithelial breast tumours. Whereas most PT showed chromosomal instability, fibroadenomas lacked copy number changes. Some copy number aberrations had not previously been associated with PT. Several well-known cancer related genes, such as TP53 and members of the Cadherin, reside within the recurrent regions of copy number alteration. Since copy number change was found in all benign PT, genomic instability may be an early event in PT genesis. Show more

Keywords: Breast, phyllodes tumour, fibroadenoma, array CGH

DOI: 10.3233/CLO-2009-0457

Citation: Analytical Cellular Pathology, vol. 31, no. 1, pp. 31-39, 2009

Price: EUR 27.50

TGF-β repressors SnoN and Ski are implicated in human colorectal carcinogenesis

Article Type: Research Article

Abstract: Background: The TGF-β signaling repressors SnoN and Ski have been critically implicated in human cancer. Methods: To explore the role of SnoN and Ski in the development and progression of colorectal cancer we examined their protein expression profile by immunohistochemistry in a series of human colorectal adenomas, carcinomas and lymph node metastases. The mRNA expression of SnoN was also quantified by Real-Time RT-PCR. Results: SnoN and Ski were overexpressed both in adenomas with severe dysplasia and colorectal carcinomas. Protein expression was cytoplasmic and nuclear with predominant cytoplasmic localization. The subcellular localization was related differently to pathologic variables of …colorectal carcinomas. Although there was no significant association of protein levels with tumor invasion and metastasis, a significant correlation of nuclear SnoN and Ski with β-catenin pathway was observed. Moreover, SnoN mRNA did not differ in carcinomas as compared to normal control and there was no correlation between SnoN protein and mRNA levels. Conclusion: Our findings suggest that SnoN and Ski exert oncogenic effects in human colorectal carcinogenesis and their overexpression is implicated in early stage disease. Show more

Keywords: Colorectal cancer, SnoN, Ski, TGF-β, β-catenin

DOI: 10.3233/CLO-2009-0460

Citation: Analytical Cellular Pathology, vol. 31, no. 1, pp. 41-51, 2009

Price: EUR 27.50