In epithelial cancers, aberrant COL17A1 promoter methylation predicts its misexpression and increased invasion

Background: Metastasis is a leading cause of death among cancer patients. In the tumor microenvironment, altered levels of extracellular matrix proteins, such as collagens, can facilitate the first steps of cancer cell metastasis, including invasion into surrounding tissue and intravasation into the blood stream. However, the degree of misexpression of collagen genes in tumors remains understudied, even though this knowledge could greatly facilitate the development of cancer treatment options aimed at preventing metastasis. Methods: We systematically evaluate the expression of all 44 collagen genes in breast cancer and assess whether their misexpression provides clinical prognostic significance. We use immunohistochemistry on 150 ductal breast cancers and 361 cervical cancers and study DNA methylation in various epithelial cancers. Results: In breast cancer, various tests show that COL4A1 and COL4A2 overexpression and COL17A1 (BP180, BPAG2) underexpression provide independent prognostic strength (HR = 1.25, 95% CI = 1.17–1.34, p = 3.03 × 10; HR = 1.18, 95% CI = 1.11–1.25, p = 8.11 × 10; HR = 0.86, 95% CI = 0.81–0.92, p = 4.57 × 10; respectively). Immunohistochemistry on ductal breast cancers confirmed that the COL17A1 protein product, collagen XVII, is underexpressed. This strongly correlates with advanced stage, increased invasion, and postmenopausal status. In contrast, immunohistochemistry on cervical tumors showed that collagen XVII is overexpressed in cervical cancer and this is associated with increased local dissemination. Interestingly, consistent with the opposed direction of misexpression in these cancers, the COL17A1 promoter is hypermethylated in breast cancer and hypomethylated in cervical cancer. We also find that the COL17A1 promoter is hypomethylated in head and neck squamous cell carcinoma, lung squamous cell carcinoma, and lung adenocarcinoma, in all of which collagen XVII overexpression has previously been shown. Conclusions: Paradoxically, collagen XVII is underexpressed in breast cancer and overexpressed in cervical and other epithelial cancers. However, the COL17A1 promoter methylation status accurately predicts both the direction of misexpression and the increased invasive nature for five out of five epithelial cancers. This implies that aberrant epigenetic control is a key driver of COL17A1 gene misexpression and tumor cell invasion. These findings have significant clinical implications, suggesting that the COL17A1 promoter methylation status can be used to predict patient outcome. Moreover, epigenetic targeting of COL17A1 could represent a novel strategy to prevent metastasis in patients

How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only ∼30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research

Selective in situ protein expression profiles correlate with distinct phenotypes of basal cell carcinoma and squamous cell carcinoma of the skin

Non-melanoma skin cancer is the most common malignancy that shows increasing incidence due to our cumulative exposure to ultraviolet irradiation. Its major subtypes, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) differ in pathobiology, phenotype and clinical behavior, which must be reflected at the molecular level. In this study, protein expression profiles of BCC and SCC were tested in tissue microarrays and correlated with that of actinic keratosis, Bowen’s disease, seborrheic keratosis and normal epidermis by detecting 22 proteins involved in cell interactions, growth, cell cycle regulation or apoptosis. The significantly more reduced collagen XVII, CD44v6, pan-Desmoglein levels and more evident E-Cadherin delocalization in BCC compared to SCC correlated with the de novo dermal invasion of BCC against the progressive invasion from in situ lesions in SCC development. EGFR was also expressed at a significantly higher level in SCC than in BCC. The upregulated cell communication protein connexin43 in BCC could contribute to the protection of BCC from metastatic invasion. Elevated cell replication in BCC was underlined by the increased topoisomerase IIa and reduced p21waf1 and p27kip1 positive cells fractions compared to SCC. Compared to differentiated keratinocytes, caspase-8 and -9 were equally upregulated in skin carcinoma subtypes for either mediating apoptosis induction or immune escape of tumor cells. Hierarchical cluster analysis grouped SCC and actinic keratosis cases exclusively together in support of their common origin and malignant phenotype. BCC cases were also clustered fully together. Differentially expressed proteins reflect the distinct pathobiology of skin carcinoma subtypes and can serve as surrogate markers in doubtful cases

Selective in situ protein expression profiles correlate with distinct phenotypes of basal cell carcinoma and squamous cell carcinoma of the skin.

Non-melanoma skin cancer is the most common malignancy that shows increasing incidence due to our cumulative exposure to ultraviolet irradiation. Its major subtypes, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) differ in pathobiology, phenotype and clinical behavior, which must be reflected at the molecular level. In this study, protein expression profiles of BCC and SCC were tested in tissue microarrays and correlated with that of actinic keratosis, Bowen's disease, seborrheic keratosis and normal epidermis by detecting 22 proteins involved in cell interactions, growth, cell cycle regulation or apoptosis. The significantly more reduced collagen XVII, CD44v6, pan-Desmoglein levels and more evident E-Cadherin delocalization in BCC compared to SCC correlated with the de novo dermal invasion of BCC against the progressive invasion from in situ lesions in SCC development. EGFR was also expressed at a significantly higher level in SCC than in BCC. The upregulated cell communication protein connexin43 in BCC could contribute to the protection of BCC from metastatic invasion. Elevated cell replication in BCC was underlined by the increased topoisomerase IIalpha and reduced p21waf1 and p27kip1 positive cells fractions compared to SCC. Compared to differentiated keratinocytes, caspase-8 and -9 were equally upregulated in skin carcinoma subtypes for either mediating apoptosis induction or immune escape of tumor cells. Hierarchical cluster analysis grouped SCC and actinic keratosis cases exclusively together in support of their common origin and malignant phenotype. BCC cases were also clustered fully together. Differentially expressed proteins reflect the distinct pathobiology of skin carcinoma subtypes and can serve as surrogate markers in doubtful cases

Collagen XVII is expressed in malignant but not in benign melanocytic tumors and it can mediate antibody induced melanoma apoptosis

The 180 kDa transmembrane collagen XVII is known to anchor undifferentiated keratinocytes to the basement membrane in hemidesmosomes while constitutively shedding a 120 kDa ectodomain. Inherited mutations or auto-antibodies targeting collagen XVII cause blistering skin disease. Collagen XVII is down-regulated in mature keratinocytes but re-expressed in skin cancer. By recently detecting collagen XVII in melanocyte hyperplasia, here we tested its expression in benign and malignant melanocytic tumors using endodomain and ectodomain selective antibodies. We found the full-length collagen XVII protein in proliferating tissue melanocytes, basal keratinocytes and squamous cell carcinoma whereas resting melanocytes were negative. Furthermore, the cell-residual 60 kDa endodomain was exclusively detected in 62/79 primary and 15/18 metastatic melanomas, 8/9 melanoma cell lines, HT199 metastatic melanoma xenografts and atypical nests in 8/63 dysplastic nevi. The rest of 19 nevi including common, blue and Spitz subtypes were also negative. In line with the defective ectodomain, sequencing of COL17A1 gene revealed aberrations in the ectodomain coding region including point mutations. Collagen XVII immunoreaction-stained spindle cell melanomas, showed partly overlapping profiles with those of S100B, Melan A and HMB45. It was concentrated at vertical melanoma fronts and statistically associated with invasive phenotype. Antibody targeting the extracellular aa507-529 terminus of collagen XVII endodomain promoted apoptosis and cell adhesion, while inhibiting proliferation in HT199 cells. These results suggest that the accumulation of collagen XVII endodomain in melanocytic tumors is associated with malignant transformation to be a potential marker of malignancy and a target for antibody-induced melanoma apoptosis. © 2012 Springer-Verlag