EMVI-positive stage II rectal cancer has similar clinical outcomes as stage III disease following pre-operative chemoradiotherapy.

BACKGROUND: Stage II rectal cancers comprise a heterogeneous group, and there is significant variability in practise with regards to adjuvant chemotherapy; the survival benefit of chemotherapy is perceived to be <4% in these patients. However, in recent years, the emergence of additional prognostic factors such as extramural venous invasion (EMVI) suggests that there may be sub-stratification of stage II tumours and, further, we may be under-estimating the benefit adjuvant chemotherapy provides in high-risk patients. This study examined the outcomes of patients with stage II and III rectal cancer to determine whether EMVI status influences disease-free survival (DFS). PATIENTS AND METHODS: An analysis of a prospectively maintained database was conducted of patients presenting with rectal cancer between 2006 and 2012. All patients underwent curative surgery and had no evidence of metastases at presentation. Clinicopathological factors were compared between stage II and III disease. The primary end point was 3-year DFS; univariate and multivariate analysis was carried out using Cox proportional hazards regression models; hazard ratios (HR) with 95% confidence intervals (CIs) were calculated. RESULTS: Four hundred and seventy-eight patients were included: 233 stage II; 245 stage III. The prevalence of EMVI was 34.9%; 57 stage II patients (24.5%) and 110 stage III patients (44.9%). On multivariate analysis, only EMVI status was a significant factor for DFS. The adjusted HR for EMVI either alone or in combination with nodal involvement was 2.08 (95% CI 1.10-2.95) and 2.74 (95% CI 1.66-4.52), respectively. CONCLUSION: EMVI is an independently poor prognostic factor for DFS for both stage II and stage III rectal cancer. These results demonstrate that there is risk-stratification within stage II tumours which affects prognosis. When discussing the use of adjuvant chemotherapy with patients that have EMVI-positive stage II tumours, these results provide evidence for a similarly increased risk of distant failure as stage III disease without venous invasion.

Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin.

BACKGROUND: Fluorescence lifetime imaging (FLIM) is a novel imaging technique that generates image contrast between different states of tissue due to differences in fluorescence decay rates. OBJECTIVES: To establish whether FLIM of skin autofluorescence can provide useful contrast between basal cell carcinomas (BCCs) and surrounding uninvolved skin. METHODS: Unstained excision biopsies of 25 BCCs were imaged en face with FLIM following excitation of autofluorescence with a 355 nm pulsed ultraviolet laser. RESULTS: Using FLIM we were able to distinguish areas of BCC from surrounding skin in an ex vivo study. Significant reductions in mean fluorescence lifetimes between areas of BCC and areas of surrounding uninvolved skin were demonstrated (P < 0.0001). These differences were apparent irrespective of the decay model used to calculate the fluorescence lifetimes (single vs. stretched exponential) or the long-pass filter through which the emitted autofluorescence was collected (375 vs. 455 nm). Conversely, there was no significant difference between the BCC and uninvolved areas of each sample when mean autofluorescence intensities were examined. Moreover, wide-field false-colour images of fluorescence lifetimes clearly discriminated areas of BCC from the surrounding uninvolved skin. CONCLUSIONS: We therefore believe that FLIM has a potential future clinical role in imaging BCCs for rapid and noninvasive tumour delineation and as an aid to determine adequate excision margins with best preservation of normal tissue.

Tumor necrosis factor and its receptors in human ovarian cancer. Potential role in disease progression.

The gene for tumor necrosis factor, TNF, was expressed in 45 out of 63 biopsies of human epithelial ovarian cancer. In serous tumors, there was a positive correlation between level of TNF expression and tumor grade. TNF mRNA was found in epithelial tumor cells and infiltrating macrophages, whereas TNF protein localized primarily to a subpopulation of macrophages within and in close proximity to tumor areas. mRNA and protein for the p55 TNF receptor gene localized to the tumor epithelium and tumor, but not to stromal macrophages. The p75 TNF receptor was confined to infiltrating cells. Cells expressing TNF mRNA were also found in ovarian cancer ascites and TNF protein was detected in some ascitic fluids. In 2 out of 12 biopsies of normal ovary, TNF mRNA was detected in a minority of cells in the thecal layer of the corpus luteum. Serum levels of TNF and its soluble receptor did not correlate with extent of TNF expression in matched biopsies. Northern and Southern analysis revealed no gross abnormality of the TNF gene. The coexpression of TNF and its receptor in ovarian cancer biopsies suggests the capacity for autocrine/paracrine action. TNF antagonists may have therapeutic potential in this malignancy.

The detection and localization of monocyte chemoattractant protein-1 (MCP-1) in human ovarian cancer.

Chemokines may control the macrophage infiltrate found in many solid tumors. In human ovarian cancer, in situ hybridization detected mRNA for the macrophage chemokine monocyte chemoattractant protein-1 (MCP-1) in 16/17 serous carcinomas, 4/4 mucinous carcinomas, 2/2 endometrioid carcinomas, and 1/3 borderline tumors. In serous tumors, mRNA expression mainly localized to the epithelial areas, as did immunoreactive MCP-1 protein. In the other tumors, both stromal and epithelial expression were seen. All tumors contained variable numbers of cells positive for the macrophage marker CD68. MCP-1 mRNA was also detected in the stroma of 5/5 normal ovaries. RT-PCR demonstrated mRNA for MCP-1 in 7/7 serous carcinomas and 6/6 ovarian cancer cell lines. MCP-1 protein was detected by ELISA in ascites from patients with ovarian cancer (mean 4.28 ng/ml) and was produced primarily by the cancer cells. Human MCP-1 protein was also detected in culture supernatants from cell lines and in ascites from human ovarian tumor xenografts which induce a peritoneal monocytosis in nude mice. We conclude that the macrophage chemoattractant MCP-1 is produced by epithelial ovarian cancer and that the tumor cells themselves are probably a major source. MCP-1 may contribute to the accumulation of tumor-associated macrophages, which may subsequently influence tumor behavior.

A hyperspectral fluorescence lifetime probe for skin cancer diagnosis.

The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and 440 nm that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected (<2 h) ex vivo skin lesions, illustrating its potential for skin cancer detection and diagnosis.

Optimal processing of bone marrow trephine biopsy: the Hammersmith Protocol.

Specimens of bone marrow trephine biopsy (BMT) are transported and fixed in acetic acid-zinc-formalin fixative, decalcified in 10% formic acid-5% formaldehyde and processed with other specimens to paraffin-wax embedding. Sections, 1-microm-thick, are cut by experienced histotechnologists and used for haematoxylin and eosin, Giemsa, reticulin silver and other histological stains. Further, all immunohistochemical procedures used in the laboratory, including double immunostaining, can be used on these sections with no or minimal modifications. About 10,000 BMT specimens have been analysed using this procedure since 1997 and diseases involving the bone marrow have been classified successfully. More recently, standardised polymerase chain reaction-based analysis and mRNA in situ hybridisation studies have been conducted. Excellent morphology with good antigen, DNA and RNA preservation is offered by the Hammersmith Protocol.

Investigation of cytokine gene expression in human colorectal cancer.

RNA was extracted from 28 samples of colorectal cancer and 26 samples of adjacent normal bowel. Northern blotting analysis showed the presence of mRNA for tumor necrosis factor (TNF) in 15 of 28 cancer samples and 6 of 26 matched normal areas. In 10 patients, TNF mRNA was found only in the tumor; in 5, TNF mRNA was seen in tumor and normal areas; and in only 1 was TNF mRNA seen in the normal, but not the malignant, area. The expression of TNF mRNA was not related to the stage of disease, degree of lymphocyte infiltration, or necrosis in the tumor. Blots were reprobed for gamma-interferon, interleukin (IL) 1 alpha and beta, IL-6, and transforming growth factor beta 1 mRNA. One tumor sample was positive for IL-1 beta, and one normal sample expressed interferon gamma mRNA. All samples had transforming growth factor beta 1 mRNA, and there was no obvious difference between levels in tumor tissues or adjacent normal areas. In situ hybridization studies with a TNF riboprobe showed that TNF mRNA was only detectable in a small minority of mononuclear and predominantly stromal cells. Immunohistochemistry on sequential sections showed that CD4- and CD8-positive lymphocytes, and macrophages, were present in the stroma. An antibody to the macrophage C3b receptor identified a minority population whose distribution corresponded closely to the cells labeled with the TNF riboprobe.

Genetic prodrug activation therapy for breast cancer: A phase I clinical trial of erbB-2-directed suicide gene expression.

PURPOSE: This trial was designed to test the safety and efficacy of a tumor-specific genetic prodrug activation therapy targeted by use of the human erbB-2 gene promoter. The erbB-2 oncogene is overexpressed in approximately 20% of cases of breast cancer and is associated with poor prognosis. PATIENTS AND METHODS: Twelve breast cancer patients received transcriptionally targeted gene therapy in a phase I clinical trial using direct intratumoral injection of plasmid construct combined with systemic administration of prodrug. The genetic prodrug activation therapy is specifically targeted to erbB-2-overexpressing breast cancer cells by use of a therapeutic cassette that contains the Escherichia coli cytosine deaminase gene driven by the tumor-specific erbB-2 promoter, thus allowing activation of fluorocytosine to the active cytotoxic fluorouracil only within tumor cells that express the oncogene. RESULTS: The approach was shown to be safe and to result in targeted gene expression in up to 90% of cases. Using a number of different assays, we demonstrated that significant levels of expression of the suicide gene were specifically restricted to erbB-2-positive tumor cells, confirming the selectivity of the approach. CONCLUSION: The results of this study, the first targeted gene therapy for breast cancer and the first to use the cytosine deaminase system in human subjects, are encouraging for the development of genetic prodrug activation therapies that exploit the transcriptional profile of cancer cells.

Toward the clinical application of time-domain fluorescence lifetime imaging.

High-speed (video-rate) fluorescence lifetime imaging (FLIM) through a flexible endoscope is reported based on gated optical image intensifier technology. The optimization and potential application of FLIM to tissue autofluorescence for clinical applications are discussed.

Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145.

A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (-508 to -501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and compatible protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer.

In situ detection of tumour necrosis factor in human ovarian cancer specimens.

In situ hybridisation was used to study the local expression of tumour necrosis factor (TNF) mRNA in human ovarian tumours. In 8 of 14 ovarian cancers studied, a minority of cells in the epithelial areas of the tumour contained TNF mRNA. In individual high-power fields as many as 8% of cells were positive for TNF mRNA. Immunohistochemical studies on sequential sections and the morphology of the positive cells led to the conclusion that the ovarian tumour cells were transcribing the TNF gene. There was immunohistochemical evidence of the production of TNF protein by the tumour cells and TNF protein in a tumour lysate. The production of TNF by human ovarian cancer cells may influence the biology of the tumour, contribute to neoplastic progression and alter the response to therapy.

Integrins and their accessory adhesion molecules in mammary carcinomas: loss of polarization in poorly differentiated tumors.

The integrins are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. To identify the pattern of expression of the beta 1, beta 3, and beta 4 integrins and their accessory adhesion molecules in relation to the malignant phenotype of invasive breast cancer, we performed an immunohistochemical study for the alpha 2 beta 1 (VLA-2), alpha 6 beta 1 (VLA-6), alpha v and alpha v beta 3 (vitronectin receptor), alpha 6 beta 4, carcinoembryonic antigen, and carcinoembryonic antigen-related molecules in a series of 37 invasive breast carcinomas. All integrin chains examined showed similar patterns in nonneoplastic breast tissue, with strong membrane staining of the myoepithelial cells and weak to moderate staining on the basolateral surfaces of the luminal cells. We found that downregulation of the alpha 2 chain of VLA-2 occurs more frequently in poorly differentiated grade III invasive ductal carcinomas (IDCs) (P = .048). Loss of alpha 6 beta 4 seems also to occur more frequently in grade III IDC (seven of 11 cases, 63.6%) than in grade I/II IDC (two of eight cases, 25%), although this did not reach statistical significance. Carcinoembryonic antigen and carcinoembryonic antigen-related antigens, which are known to function as accessory adhesion molecules, were found mainly in the cytoplasm of neoplastic cells and there was reduced membrane polarization in poorly organized tumors. In contrast the alpha v beta 3, vitronectin receptor heterodimer recognized by the 23C6 monoclonal antibody was weak or absent in normal breast epithelium, and was weakly expressed in two of 19 (10%) IDCs and in nine of 18 (50%) invasive lobular carcinomas (P = .008). However, the alpha v chain detected with the antibody 13C2 was weakly to moderately expressed on nonneoplastic epithelium and at a similar intensity in 13 of 19 IDCs and 15 of 17 invasive lobular carcinomas, suggesting that in IDC the alpha v chain may be associated with a different beta chain (possibly beta 1 or beta 5). No correlation between integrin expression and estrogen/progesterone receptor status was found. These data provide further evidence that in invasive breast carcinomas there is a widespread deregulated expression of integrins and their accessory adhesion molecules with loss of polarization. Changes in the expression and function of cell adhesion molecules, which control growth and differentiation, may have clinical relevance in the behavior of breast cancer.

Characterization of monoclonal antibodies raised to C-terminal peptides of pS2: a major trefoil peptide and motility factor expressed in adenocarcinomas and regions of mucosal injury.

Two novel monoclonal antibodies, GE1 and GE2 raised against the C-terminal 31 and 28 amino acids of the estrogen-inducible trefoil peptide pS2, are described. Both antibodies are able to detect pS2 in formalin-fixed, paraffin-embedded tissues. Conditions are presented under which pS2 can be shown in cell lines by immunohistochemistry that has previously been problematic. The antibodies can specifically show the presence of pS2 in cell lysates by Western blotting and immunoprecipitation. In the form of an affinity column, the GE1 monoclonal antibody can be used to purify pS2 from MCF-7 supernatants. The eluted peptide from the GE1 affinity column shows a single band at 6,600 Da (predicted size for pS2) on Western blotting. These antibodies are valuable reagents in the analysis of the role of trefoil peptides in the maintenance of mucosal integrity, and may have applications in the assessment of pS2 expression in chronic gastrointestinal ulceration and adenocarcinomas that secrete pS2, where it may serve as a prognostic marker.

MT1-MMP and MMP-2 mRNA expression in human ovarian tumors: possible implications for the role of desmoplastic fibroblasts.

Expression of activated MMP-2 (72 kDa type IV collagenase) is highly associated with the malignant phenotype in adenocarcinomas, but predominant expression of the mRNA appears to be in stromal cells. MT1-MMP (membrane type 1-matrix metalloproteinase) is implicated in tumor-epithelial cell surface activation of latent pro-MMP-2, indicating a mechanism for tumor-stromal interaction in invasion. We determined the relative mRNA distribution of these MMPs in human ovarian tumors with a view to analyzing potential variations in the epithelial-mesenchymal interactions dictating ovarian tumor cell spread. In situ hybridization using 35S-labeled riboprobes was used to analyze 33 human ovarian tumors and mouse xenografts of human ovarian (DOV 13, SKOV3) and breast (MCF 7) tumor cell lines known to express MT1-MMP and MMP-2. MMP-2 mRNA was expressed in 31 of 33 and MT1-MMP mRNA was expressed in 29 of 33 tumor cases. MMP-2 mRNA was predominantly expressed in desmoplastic fibroblasts and in the subepithelial stroma. MT1-MMP mRNA showed some colocalization with MMP-2 in stromal cells. Neoplastic epithelial cell labeling for MT1-MMP mRNA was present in borderline and malignant tumors but not in benign tumors, and was invariably less than stromal labeling. Xenografts of DOV 13, SKOV 3, and MCF 7 cells showed some stromal localization of MMP-2 mRNA and weak labeling of DOV 13 cells. There was variable labeling for MT1-MMP mRNA in the neoplastic cells only. The colocalization of MT1-MMP and MMP-2 mRNAs in ovarian carcinoma stroma supports the view that MT1-MMP is closely associated with MMP-2 expression and function. It suggests that either additional mechanisms are involved in regulating MMP-2 activation at the tumor cell surface, or more intriguingly, that desmoplastic fibroblasts may be the primary mediators of extracellular matrix remodeling with respect to this system.

Accumulation of ceroid in smooth muscle indicates severe malabsorption and vitamin E deficiency.

Four patients had accumulation of ceroid in smooth muscle (lipofuscinosis), which indicated severe or uncontrolled malabsorption, with confirmed vitamin E deficiency in three cases. The distribution of the pigment was systematic, and there seemed to be an association between malabsorption syndrome and vitamin E deficiency. Vitamin E supplementation seems to be indicated in such patients, and it is suggested that studies of smooth muscle function should be made in cases of heavy accumulation of ceroid.

Antral hypertrophic gastritis: a rare cause of iron deficiency.

A case of an unusual hypertrophic gastropathy confined to the gastric antrum which presented with chronic anaemia is described. The clinical and pathological features are contrasted with Menetrier's disease and hypersecretory hypertrophic gastropathy, and a possible relation to solitary hyperplastic (regenerative) polyps is discussed.

Expression of gelatinase A and TIMP-2 mRNAs in desmoplastic fibroblasts in both mammary carcinomas and basal cell carcinomas of the skin.

AIMS: To compare the localisation of mRNAs for the basement membrane degrading enzyme gelatinase A (72 kilodalton type IV collagenase) and its inhibitor TIMP-2 in carcinomas of the breast and basal cell carcinomas of the skin which have little or no ability to metastasize. METHODS: In situ hybridisation was performed on formalin fixed, paraffin wax embedded blocks using 35S-labelled riboprobes on 16 mammary carcinomas, three fibroadenomas, and a benign phyllodes tumour, and on 15 basal cell carcinomas of the skin (BCC). RESULTS: Labelling for both mRNAs was detectable in 14 of 16 mammary carcinomas and in 13 of 15 BCC, most often over organising desmoplastic fibroblasts in the stroma around invasive epithelial aggregates. Some sparse labelling was seen over malignant epithelial cells in six of the mammary carcinomas but not in the BCC. Some expression of gelatinase A mRNA was also seen in fibroblasts of breast lobules adjacent to the mammary carcinomas and around engulfed adnexal elements in the BCC, but not in unaffected breast tissues, fibroadenomas, the phyllodes tumour or unaffected skin. CONCLUSIONS: Maximal expression of gelatinase A and TIMP-2 mRNAs occurs in malignant neoplasms as part of the host response to the presence of established neoplastic cells rather than as an initial response to invasion. The degree to which this is present suggests this may be a highly relevant mechanism modulating tumour differentiation, growth and progression, possibly entailing uptake via specific receptors on the tumour cell surface.

Changes in E-cadherin immunoreactivity in the adenoma-carcinoma sequence of the large bowel.

We have used an avidin-biotin immunoperoxidase technique to localise epithelial cadherin (E-cadherin), a calcium-dependent cell-cell adhesion molecule, in 107 paraffin-embedded sections from 93 patients consisting of 24 with colorectal adenoma, 55 with rectal carcinoma and 14 with liver metastases. The corresponding primary colorectal tumours were also studied in these cases. E-cadherin was expressed by normal colorectal epithelial cells with typical membranous staining at the intercellular junctions. Loss of normal membranous E-cadherin expression and presence of cytoplasmic staining were found frequently in adenomas larger than 1 cm (P < 0.01), with high grade dysplasia and villous histology (P < 0.01). In primary rectal cancers, loss of membranous expression correlated with high tumour grade. No correlation was seen with Dukes and Jass stage, local extramural spread and 5-year recurrence rate. Complete loss of membranous E-cadherin immunoreactivity was seen in 7/14 (50%) liver metastases in which 6/7 (86%) showed intense membranous E-cadherin immunoreactivity in the corresponding primary tumour. Our data indicate that changes in E-cadherin immunoreactivity and cellular localisation correlate with size, severe dysplasia in adenomas and tumour grade in carcinomas. However, there seems to be no correlation between loss of membranous E-cadherin immunoreactivity and the invasive and metastatic potential of the carcinomas.

Correlation between androgen receptor expression and FGF8 mRNA levels in patients with prostate cancer and benign prostatic hypertrophy.

AIM: To investigate the correlation between androgen receptor expression and fibroblast growth factor 8 (FGF8) mRNA levels. METHODS: 39 human prostate cancers and 14 benign prostatic hypertrophy specimens were examined immunohistochemically for androgen receptor expression and by in situ hybridisation and reverse transcription polymerase chain reaction for FGF8 expression. RESULTS: In 39 tumours there was a statistically significant negative correlation between tumour grade and FGF8 expression and a positive correlation between FGF8 and androgen receptor expression. All 14 benign hypertrophy specimens expressed moderate to high levels of FGF8 and androgen receptor. CONCLUSIONS: Loss of FGF8 may be a factor involved in the development of prostatic cancer.