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ABSTRACT: Immunohistochemical staining for FHIT and PCNA proteins was carried out in 451 breast lesions showing nonproliferative benign breast disease (BBD) (n=263), proliferative BBD without atypia (n=128), proliferative BBD with atypia (n=11), carcinoma in situ (n=15) or invasive carcinoma (n=34) and for EGFR protein in a subset of 71 of these cases. FHIT underexpression was not detected in nonproliferative lesions, but occurred in 2% of proliferative BBD without atypia, 10% proliferative BBD with atypia, 27% of carcinoma in situ and 41% of invasive carcinoma, which suggests that it could be useful in assessing those carcinoma in situ lesions (ductal, DCIS and lobular, LCIS) that are more likely to progress to malignancy. Preliminary microarray comparisons on DCIS and invasive carcinoma samples dissected from formalin-fixed paraffin sections showed a consistent downregulation of two previously identified FHIT-related genes, caspase 1 and BRCA1 in lesions underexpressing FHIT.
British Journal of Cancer 02/2007; 96(1):110-7. · 5.04 Impact Factor
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R Roylance,
P Gorman,
T Papior,
Y-L Wan,
M Ives,
J E Watson,
C Collins,
N Wortham,
C Langford,
H Fiegler,
N Carter,
C Gillett,
P Sasieni,
S Pinder, A Hanby,
I Tomlinson
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ABSTRACT: We analysed chromosome 16q in 106 breast cancers using tiling-path array-comparative genomic hybridization (aCGH). About 80% of ductal cancers (IDCs) and all lobular cancers (ILCs) lost at least part of 16q. Grade I (GI) IDCs and ILCs often lost the whole chromosome arm. Grade II (GII) and grade III (GIII) IDCs showed less frequent whole-arm loss, but often had complex changes, typically small regions of gain together with larger regions of loss. The boundaries of gains/losses tended to cluster, common sites being 54.5-55.5 Mb and 57.4-58.8 Mb. Overall, the peak frequency of loss (83% cancers) occurred at 61.9-62.9 Mb. We also found several 'minimal' regions of loss/gain. However, no mutations in candidate genes (TRADD, CDH5, CDH8 and CDH11) were detected. Cluster analysis based on copy number changes identified a large group of cancers that had lost most of 16q, and two smaller groups (one with few changes, one with a tendency to show copy number gain). Although all morphological types occurred in each cluster group, IDCs (especially GII/GIII) were relatively overrepresented in the smaller groups. Cluster groups were not independently associated with survival. Use of tiling-path aCGH prompted re-evaluation of the hypothetical pathways of breast carcinogenesis. ILCs have the simplest changes on 16q and probably diverge from the IDC lineage close to the stage of 16q loss. Higher-grade IDCs probably develop from low-grade lesions in most cases, but there remains evidence that some GII/GIII IDCs arise without a GI precursor.
Oncogene 11/2006; 25(49):6544-53. · 6.37 Impact Factor
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C Palmieri,
D Roberts-Clark,
A Assadi-Sabet,
R C Coope,
M O'Hare,
A Sunters, A Hanby,
M J Slade,
J J Gomm,
E W-F Lam,
R C Coombes
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ABSTRACT: Keratinocyte growth factor/fibroblast growth factor 7 (KGF/FGF7) is known to be a potent growth factor for mammary cells but its origin, cellular targets and mode of action in the breast are unclear. In this study, we carried out studies to determine the localisation of FGF7 and its receptor, and the related growth factor FGF10. We also determined the factors that regulate FGF7 release from stromal cells and the effects of FGF7 on normal and neoplastic breast cells. Using an FGF7-specific antibody which does not react with the FGF7 heparan sulphate proteoglycan (HSPG)-binding site, we showed epithelial and myoepithelial immunohistochemical staining in normal breast sections, and epithelial staining in breast carcinomas. Stromal staining was also detected in some lobular carcinomas as well as a subset of invasive ductal carcinomas. FGF10 and FGF receptor (FGFR)2 immunostaining showed a similar epithelial expression pattern, whereas no stromal staining was observed. We purified normal breast stromal, epithelial and myoepithelial cells and showed that FGF7 stimulated proliferation of both epithelial cell types, but not stromal fibroblasts. We also examined the effects of FGF7 on Matrigel-embedded organoids, containing both epithelial and myoepithelial cells, and showed FGF7 induced an increase in cellular proliferation. Furthermore, conditioned medium derived from stromal cells was shown to increase the proliferation of normal and neoplastic breast epithelial cells, which could be abolished by a neutralising antibody to FGF7. Finally, we showed that interleukin-1beta, but not oestradiol or other oestrogen receptor ligands, caused a dose-related FGF7 release. Further results also indicate that the epithelial localisation of FGF7 and FGF10 in breast tissue sections is likely to be due to their binding to their cognate receptor. In summary, our findings suggest that FGF7 is a paracrine growth factor in the breast. FGF7 is produced by the breast stromal fibroblasts and has profound proliferative and morphogenic roles on both epithelial and myoepithelial cells.
Journal of Endocrinology 05/2003; 177(1):65-81. · 3.55 Impact Factor
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ABSTRACT: The cell-cell adhesion receptor gene E-cadherin (CDH1) is expressed by epithelial cells, in which it mediates adhesion and morphogenesis. Invasive lobular carcinoma (ILC) characteristically infiltrates diffusely as single cells; by immunohistochemistry, many of these tumours lack E-cadherin expression. In the present study we investigated various ways in which loss of function of the E-cadherin gene could occur in ILCs, namely, promoter methylation, mutation and allelic loss. We analysed 22 ILCs and found 12 (55%) E-cadherin-negative samples by immunohistochemical analysis. Methylation-specific polymerase chain reaction (PCR) showed that 17/22 (77%) of these tumours had methylation of the CDH1 promoter, including 11/12 (91%) of the E-cadherin-negative tumours. All 16 exons of E-cadherin (including intron-exon boundaries) were amplified from chromosomal DNA and screened for mutations by conformation-sensitive gel electrophoresis (CSGE). Bands with altered mobility were analysed by direct sequencing. We identified five frameshift mutations, which resulted in downstream stop codons and one splice site mutation in six different tumours (29%). Loss of heterozygosity (LOH) was assessed using microsatellite markers, and 9/18 (50%) informative tumours showed LOH. We conclude that most ILCs show genetic or epigenetic changes affecting the E-cadherin gene and that many of these tumours lack E-cadherin expression. In all cases in which there was loss of expression, this was consistent with biallelic inactivation of CDH1 by promoter methylation, mutation or allelic loss in any combination.
International Journal of Cancer 06/2001; 92(3):404-8. · 5.44 Impact Factor
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ABSTRACT: To investigate whether expression of E-cadherin correlates with polarised tissue organisation, grade or tumour type in salivary neoplasms, frozen sections from 30 salivary gland neoplasms were stained immunohistochemically for E-cadherin using the antibody HECD-1 and compared to the staining patterns in five samples of normal salivary gland. Lesions with areas of lack of staining were restained at two higher antibody concentrations. Normal salivary gland stained strongly around the periphery of acinar and ductal cells. Neoplasms mostly stained strongly regardless of neoplasm type. Reproducible loss of expression was found only in epithelial cells showing stromal or plasmacytoid (hyaline) differentiation in pleomorphic adenoma. Low- and high- grade mucoepidermoid carcinomas, adenocarcinoma NOS and carcinoma ex pleomorphic adenoma showed focal loss of expression but this was not related to tissue architecture, differentiation or invasiveness. We conclude that the relationship seen between E-cadherin expression and cell polarity/glandular organisation in breast and colon does not appear to exist for salivary gland neoplasms in which the diversity of architectural patterns precludes detection of any simple relationship. E-cadherin expression seems unlikely to be a useful marker for diagnosis or prognosis in salivary neoplasia in general.
Oral Oncology 12/2000; 36(6):515-8. · 2.86 Impact Factor
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ABSTRACT: The urokinase-type plasminogen activator (uPA) system has been implicated in tumor spread. We have used immunohistochemistry to examine three components of this system, ie, uPA, uPA receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1), in a pilot study on 142 cases of breast carcinoma. We wished to determine whether there were any relationships between expression of the proteins in either tumor cells or fibroblasts and clinical and pathological features. Strong uPA expression in each cell type was significantly related to high tumor grade (P = 0.013 and 0.008, respectively), and was more common in invasive than in in situ carcinomas (P < 0.0001). Fibroblastic expression of uPAR was only related to the presence of invasion (P < 0.0001). Strong PAI-1 expression in both cell types was seen in high-grade tumors (tumor cells, P = 0.012; fibroblasts, P < 0.001), but only fibroblastic expression was related to the presence of invasion (P = 0.042). Fibroblastic expression of both uPA and uPAR were positively correlated with tumor size. Although patients with strong fibroblastic expression of uPA showed a tendency toward a shorter time to relapse, none of the plasminogen activator proteins were significantly associated with relapse-free survival. These results suggest that strong expression of uPA, uPAR, and PAI-1 in fibroblasts rather than in tumor cells have the most impact on the clinical behavior of breast cancer. Larger prospective studies are needed to confirm these findings.
American Journal Of Pathology 11/2000; 157(4):1219-27. · 4.89 Impact Factor
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ABSTRACT: The LKB1/STK11 serine/threonine kinase is mutated in Peutz-Jeghers' syndrome and acts as a tumour suppressor. Using northern blotting and RT-PCR, LKB1 has been reported to be expressed widely in human adult tissues, although in Xenopus the expression of its homologue, XEEK1, is apparently restricted to early embryogenesis. In situ hybridization has been used to detect and localize LKB1 mRNA in a variety of adult and fetal tissues and tumours. The results show that LKB1 expression is widespread, but predominant in epithelia and in the seminiferous tubules of the testis. Expression is higher in fetal than in adult tissues. Expression also appears to be higher in many malignant tumours than in normal tissues or benign lesions, although some cancers have lost LKB1 expression, quite possibly as part of the process of tumourigenesis. These data are consistent with a widespread functional role for LKB1 in tissues of most types, and with a role for LKB1 in the pathogenesis of some sporadic cancers. LKB1 expression may primarily be related to the rate of cell replication.
The Journal of Pathology 11/2000; 192(2):203-6. · 6.32 Impact Factor
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ABSTRACT: It is not clear whether APC mutations are sufficient for early colorectal adenomas to grow or whether additional mutations at other loci are required. We previously have screened 210 early colorectal adenomas from familial adenomatous polyposis patients for mutations and allelic loss at APC. Here, we determined whether allelic loss at APC had any effect on the nearby alpha-catenin gene. However, loss on 5q in familial adenomatous polyposis adenomas rarely extended as far as alpha-catenin, and no differences in alpha-catenin protein expression were found in tumors that showed loss encompassing both APC and alpha-catenin. We then screened all 210 tumors for mutations at candidate loci other than APC (K-ras, beta-catenin, and allelic loss at 1p33-p35 and 1p36) and for microsatellite instability (MSI). Each of these loci has been implicated previously in early colorectal tumorigenesis. One tumor harbored a beta-catenin mutation and another MSI, but none showed K-ras mutation or allelic loss at 1p33-p35 or 1p36. These data support the following hypotheses derived from sporadic colorectal tumors: beta-catenin mutations are generally an alternative to mutations at APC, MSI is not usually an early phenomenon in colorectal tumorigenesis, and K-ras mutations are more typical of large- and moderate-sized adenomas. Contrary to some previous reports, chromosome 1p allelic loss is infrequent in very early adenomas. APC mutations are generally sufficient for colorectal tumors to grow to about 1-cm diameter, although chance mutations at other loci can provide these early colorectal adenomas with a selective advantage, and some colorectal tumors may develop along a pathway not involving APC.
Proceedings of the National Academy of Sciences 03/2000; 97(5):2225-8. · 9.68 Impact Factor
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ABSTRACT: How does breast cancer progress? There is evidence both to support (S. W. Duffy et al., Br. J. Cancer, 64: 1133-1138, 1991; R. Rajakariar et al., Br. J. Cancer, 71: 150-154, 1995) and refute (M. Hakama et al., Lancet, 345: 221-224, 1995; R. R. Millis et al., Eur. J. Cancer, 34: 548-553, 1998) the hypothesis of dedifferentiation; the theory that as breast cancers grow they evolve from well differentiated (grade I) to poorly differentiated (grade III) tumors. We provide evidence to support the view that the majority of grade I tumors do not progress to grade III tumors. Comparative genomic hybridization was used to screen entire genomes of a large sample (40 grade I and 50 grade III) of invasive ductal breast carcinomas, stratified by grade. We found distinct genetic differences between grade I and grade III tumors. Significantly, we found that 65% of grade I tumors lost the long arm of chromosome 16 compared with only 16% of grade III tumors. This pattern of loss leads us to conclude that the majority of grade I tumors do not progress to grade III tumors. These findings have important implications because they suggest that different breast tumor grades may have distinct molecular origins, pathogenesis, and behavior and, therefore, potentially present distinct molecular targets for research and treatment.
Cancer Research 05/1999; 59(7):1433-6. · 7.86 Impact Factor
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T Ng,
A Squire,
G Hansra,
F Bornancin,
C Prevostel, A Hanby,
W Harris,
D Barnes,
S Schmidt,
H Mellor,
P I Bastiaens,
P J Parker
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ABSTRACT: Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.
Science 04/1999; 283(5410):2085-9. · 31.20 Impact Factor
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ABSTRACT: To examine the value of immunohistochemistry in defining a keratin profile to aid cervical histopathological diagnosis.
Immunohistochemical localisation of keratins 17, 10, and 19 was studied in 268 cervical biopsies from 216 women including normal epithelia (with and without human papilloma virus), low and high grade cervical intraepithelial neoplasia, and invasive carcinoma. The percentage of positive immunostaining was scored using a Kontron MOP videoplan image analyser.
All major categories of cervical epithelia expressed these keratins to varying degrees. The median percentage of immunostaining for keratin 10 was 40% in normal tissue compared with just 1% in invasive carcinoma (p < 0.0001). The medians for keratin 17 were 0% in the normal group and 80% in carcinomas (p < 0.0001). By contrast, there was no significant difference in staining for keratin 19. Using a combination of the keratin 10 and 17 percentages, it was possible to separate the carcinomas from the benign conditions with a sensitivity of 100% and a specificity of 93%. Further analyses within the groups revealed more extensive staining for keratins 10 and 19 in reserve cell hyperplasia, immature squamous metaplasia, and congenital transformation zone.
The morphological variety within the cervix is reflected, in part, by distinct keratin patterns. There are striking differences in the patterns of keratins 10 and 17 between infiltrating squamous carcinoma and normal cervical epithelia.
Journal of Clinical Pathology 02/1999; 52(1):41-6. · 2.31 Impact Factor
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ABSTRACT: Changes in integrin expression have been shown to be important for the growth and metastatic capacity of melanoma cells. In this study, we have examined the expression of alphav integrins by three uveal and four cutaneous malanoma lines. No lines expressed alphavbeta6 and only TXM13, a cutaneous line, expressed alphavbeta8. All lines expressed alphavbeta5 and alphavbeta3 (four out of four cutaneous, two out of three uveal) or avpl (OM431, an uveal line). Thus, OM431 is the second uveal melanoma we have described that expresses alphavbeta1 and this, we report again, functions as an alternative vitronectin/fibronectin receptor. Subcutaneous growth of cell lines in athymic mice correlated with an alphavbeta3-positive, alphavbeta1 -negative phenotype. Analysis of clinical material from cutaneous melanoma showed that although alphav expression was increased in 88% of metastases, this could not all be explained by up-regulation of alphavbeta3, with only 2 out of eight skin metastases expressing this heterodimer. Using antibody SZ.21, which as we report here works in archival material, only 1 out of 15 uveal metastases expressed detectable beta3. Thus, acquisition of alphavbeta3 expression, which has been implicated in cutaneous melanoma progression, may not be required for development of metastases from uveal melanoma or indeed for skin, as distinct from lymph node, metastases of cutaneous melanoma.
British Journal of Cancer 03/1998; 77(4):522-9. · 5.04 Impact Factor
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ABSTRACT: Stromelysin 3 (ST3) is a matrix metalloproteinase implicated in mammary carcinoma progression. To date, localization of ST3 expression in breast cancer by in situ hybridization and immunocytochemistry has shown that the expression of the enzyme is limited to only the stromal fibroblasts surrounding the cancer cells. We have immunostained a large group of ductal carcinoma in situ and invasive breast carcinomas using a monoclonal antibody (5ST-4A9) raised against the hemopexin-like domain of human ST3. We show that invasive lobular carcinomas express significantly less ST3 than invasive ductal carcinomas (IDCs) (P = 0.002). We also show, for the first time, that certain breast carcinoma cells that have undergone a degree of epithelial-to-mesenchymal transition, the so-called metaplastic carcinomas, can express ST3 mRNA and protein, which may in part explain the increased metastatic propensity seen in a number of these tumors. In addition, patients with IDC who had moderate to strong ST3 levels had significantly shorter disease-free survival than those with negative or weak ST3 levels (P = 0.02). Furthermore, in node-positive IDC patients, multivariate analysis revealed that ST3 level was a strong, independent prognostic parameter for disease-free survival (P = 0.005).
American Journal Of Pathology 03/1998; 152(3):721-8. · 4.89 Impact Factor
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ABSTRACT: Loss of E-cadherin expression has been implicated in the development of invasive characteristics in colorectal carcinomas. Failure to express E-cadherin may result from mutations of the E-cadherin gene (HSECAD).
To examine the correlation between E-cadherin expression and genetic changes at HSECAD; and to examine differences in E-cadherin expression and genetic changes at HSECAD between three different groups of colorectal cancer--replication error positive (RER+) sporadic cancers, RER--sporadic cancers and ulcerative colitis associated cancers.
Sixty eight colorectal cancers (22 RER+ sporadic cancers, 32 RER- sporadic cancers and 14 ulcerative colitis associated cancers) were studied using immunohistochemistry and for allele loss at the HSECAD locus. Exon 16 of HSECAD contains several mononucleotide repeat tracts which are very similar to microsatellite repeats and which may be susceptible to replication errors (manifest as new alleles). All cases were also examined for new alleles in exon 16.
Absent or decreased E-cadherin protein expression was found in 27 (38%) of 68 colorectal cancers and the pattern of expression did not differ significantly among the three tumour groups. Allele loss occurred at HSECAD in four (10%) of 40 informative cancers and there were no differences between the three subgroups. New alleles at exon 16 were detected in three (14%) of 22 RER+ tumours; no new alleles were detected in RER- or ulcerative colitis associated cancers. Overall, there was no correlation between allelic loss or exon 16 replication errors and immunohistochemical E-cadherin expression.
(1) Loss of E-cadherin expression probably does not occur as a result of mutation at the HSECAD locus in colorectal cancers. (2) There is no difference in the frequency of loss of heterozygosity at HSECAD among RER+, RER- and ulcerative colitis associated colorectal cancers.
Gut 06/1997; 40(5):654-9. · 10.11 Impact Factor
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ABSTRACT: Defects in mismatch repair (MMR) can result in the development of a 'mutator phenotype', manifest as an increase in DNA replication errors (RERs). Patients with hereditary non-polyposis colorectal cancer (HNPCC) have germline mutations in MMR genes. These patients develop carcinomas of the colon and other specific sites at a significantly earlier age than patients with sporadic carcinomas. RERs are found in the cancers from patients with HNPCC and have been demonstrated in 10-20 per cent of sporadic colorectal cancers (CRCs). Loss of MMR may simply accelerate tumour development, but it is also possible that these tumours follow a different carcinogenetic pathway from tumours with intact MMR. In particular, it has been suggested that p53 mutations occur less often in RER-positive (RER+) sporadic colorectal cancers. In this study, the clinico-pathological features and frequency of p53 overexpression in 17 left-sided RER+ CRCs were compared with 35 left-sided RER- CRCs. No differences were found in the age and tumour stage at presentation, mucinous differentiation, or Jass prognostic grouping between these two types of CRC. Thirteen out of 17 (76 per cent) RER+ and 19/35 (54 per cent) RER- tumours showed overexpression of p53, a non-significant difference (chi 2 test). Although some previous studies have suggested differences in the clinico-pathological features and p53 expression of RER+ and RER- right-sided CRCs, our results show that these differences do not exist in left-sided cancers.
The Journal of Pathology 09/1996; 179(4):370-5. · 6.32 Impact Factor
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ABSTRACT: Several studies have examined the role of tests for human papillomavirus (HPV) in screening for cervical cancer but as yet the relevance is unclear. We looked at HPV testing for types 16, 18, 31, and 33 on material taken at the time of a cervical smear in 2009 eligible women having routine screening. Women with any degree of dyskaryosis or high levels of one of these HPV types were referred for colposcopy. 44% of the cervical intraepithelial neoplasia (CIN) lesions of grade 2/3 detected had negative cytology and were found only by HPV testing. A further 22% of the CIN 2/3 lesions were positive for HPV but showed only borderline or mild cytological changes. The positive predictive value of HPV testing was 42%, which was similar to that for moderate dyskaryosis. HPV types 16 and 31 were more sensitive and specific for CIN 2/3 than were types 18 or 33. However, 25% of the CIN 2/3 lesions were not detected by these four HPV tests. We suggest that HPV testing could usefully augment but not replace conventional cytology. These results should stimulate a much larger randomised trial to assess the impact of these improved CIN 2/3 detection rates on the subsequent incidence of invasive cancer.
The Lancet 07/1995; 345(8964):1533-6. · 38.28 Impact Factor
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ABSTRACT: In this study we show that heterologous expression of annexin VI in A431 squamous carcinoma cells caused a marked suppression of tumour cell growth when cells were cultured subcutaneously in nude mice. The tumours formed by the annexin VI+ A431 cells were morphologically and histologically similar to those formed by the wild-type cells.
British Journal of Cancer 05/1995; 71(4):786-8. · 5.04 Impact Factor
