Modification of gene expression induced by siRNA targeting of estrogen receptor alpha in MCF7 human breast cancer cells.

To establish a model of endocrine resistant breast cancer that is associated with loss of estrogen receptor (ER), MCF7 cells were transfected with several plasmid constructs intended to produce intracellular double stranded hairpin RNA to be processed into siRNA directed against different regions of the ERalpha mRNA. Stably transformed cells were propagated in long-term culture. One of these lines, designated pII, was selected for further analysis. pII cells exhibited reduced levels of ERalpha mRNA and protein as well as several estrogen-regulated genes assessed by real-time PCR and were unresponsive to addition of estradiol and tamoxifen. Higher levels of ERbeta were measurable as compared with parental MCF7 cells. There was an unexpected decrease in expression in members of the EGFR family in contrast with observations reported for ER-negative tumours or some other established endocrine-independent lines. Microarray gene analysis comparing expression in parental MCF7 with pII cells in both serum-synchronised and non-synchronised conditions highlighted a spectrum of other genes that were expressed at different levels compared to the parental MCF7 cells. Genes showing the greatest change were mostly common between synchronized and unsynchronised cells; GRB7, PSMD7, KRT19, KRT18, AKT1, SYNCRIP, CYB5A and EVL for down-regulated in pII and QDPR, VIM, CD68, CA9, STMN1, CDK2, CTSC for up-regulated in pII cells. Notably, the decreased expression of epithelial keratins 18 and 19 and an increase in vimentin and in a macrophage marker CD68, is suggestive of an epithelial to mesothelial transition. Further characterisation of these cells particularly with respect to the factors controlling their growth may contribute to a better understanding of the behaviour of cells that have become endocrine independent by loss of ER function.

Mechanisms of drug resistance in cancer chemotherapy.

The management of cancer involves procedures, which include surgery, radiotherapy and chemotherapy. Development of chemoresistance is a persistent problem during the treatment of local and disseminated disease. A plethora of cytotoxic drugs that selectively, but not exclusively, target actively proliferating cells include such diverse groups as DNA alkylating agents, antimetabolites, intercalating agents and mitotic inhibitors. Resistance constitutes a lack of response to drug-induced tumour growth inhibition; it may be inherent in a subpopulation of heterogeneous cancer cells or be acquired as a cellular response to drug exposure. Resistance varies. Although regulatory approval may require efficacy in as few as 20% of trial cohorts, a drug may subsequently be used in unselected patients displaying resistance to the treatment. Principal mechanisms may include altered membrane transport involving the P-glycoprotein product of the multidrug resistance (MDR) gene as well as other associated proteins, altered target enzyme (e.g. mutated topoisomerase II), decreased drug activation, increased drug degradation due to altered expression of drug-metabolising enzymes, drug inactivation due to conjugation with increased glutathione, subcellular redistribution, drug interaction, enhanced DNA repair and failure to apoptose as a result of mutated cell cycle proteins such as p53. Attempts to overcome resistance mainly involve the use of combination drug therapy using different classes of drugs with minimally overlapping toxicities to allow maximal dosages and with narrowest cycle intervals, necessary for bone marrow recovery. Adjuvant therapy with P-glycoprotein inhibitors and, in specific instances, the use of growth factor and protein kinase C inhibitors are newer experimental approaches that may also prove effective in abrogating or delaying onset of resistance. Gene knockout using antisense molecules may be another effective way of blocking drug resistance genes. Conversely, drug resistance may also be used to good purpose by transplanting retrovirally transformed CD34 cells expressing the MDR gene to protect the bone marrow during high-dose chemotherapy.

Allelic variation of BAT-25 and BAT-26 mononucleotide repeat loci in tumours from a group of young women with breast cancer.

Genomic instability in the form of repeat number variation at microsatellite loci has been reported in several human cancers. To resolve current confusion regarding frequency of microsatellite instability (MSI), standardised protocols have proposed use of a consensus set of informative loci; it is claimed that analysis of 2 apparently quasi-homozygous, quasi-monomorphic, mononucleotide repeats (BAT-25 and BAT-26) is sufficiently accurate to define MSI (obviating need for corresponding constitutive DNA). We examined these loci in 163 breast cancers, 108 of which had previously been analysed at 11-24 other loci and found to have MSI in 38% of them. For BAT-26 only 1/153 (homozygous) tumours showed a contracted allele, with minor size variations (1-6 bp) between individuals. For BAT-25 repeat contractions were unambiguously observed in 12 (7.4%) tumours; only 4 of these were previously designated MSI+. DNA from normal individuals showed significant allelic variation in 8/159 (5%) cases for BAT-25; no instance of heterozygosity was seen for BAT-26. Subsequently, we analysed normal DNA from the 12 individuals whose tumours had MSI at BAT-25; in 2 cases there was germline heterozygosity. We conclude that analysis with BAT-26 (in contrast to other loci) was not a useful detector of MSI. With BAT-25, a low frequency of MSI not much greater than germline polymorphism, also limits the utility of this marker for determining MSI in breast cancer.

Family history of benign thyroid disease and cancer and risk of thyroid cancer.

In a population-based study of 313 case-control pairs in Kuwait, we evaluated whether a family history of benign thyroid disease (BTD) and thyroid or other cancers was associated with an increased risk of thyroid cancer, the second most common neoplasm among women in this and several other Arab countries in the Gulf region. Family history of BTD was reported by 78 (24.9%) cases and 40 (12.8%) controls in 132 and 57 relatives, respectively. There was an approximately 2-fold increased risk of thyroid cancer in individuals who had a mother (Odds Ratio (OR)=2.3; 95% Confidence Intervals (95% CI): 1.1-5.1), sister(s) (OR=2.6; 95% CI: 1.3-5.3) or aunt(s) (OR=2.1; 95% CI: 0.9-5.3) with BTD; there was also a significant trend in increasing risk with an increasing number of affected female relatives (P<0.0001). Stratification by age at diagnosis of the case showed that individuals aged

Frequent microsatellite alterations in a predominantly younger age of onset breast cancer population.

Microsatellite instability (MSI) and loss of heterozygosity (LOH) was investigated in paired tumour and normal tissue DNA from 108 predominantly premenopausal breast cancer patients (under age 45 years at presentation) for 25 simple repeat loci interspersed across 11 chromosomes. MSI was observed at a single locus in 69 (64%) patients; 41 of these had instability at more than one site. Greatest frequency of MSI was at loci D2S1356 (33%), D2S2739 (22%), D3S1766 (21%) and D13S796 (20%). LOH was seen at a single site in 55% of patients and at two or more sites in 27 patients with greatest frequency at D2S1356 (33%), D2S443 (19%) and D17S1299 (18%). Both mutations were found in the same patient but at different loci. Clearly, choice of loci is a determining factor in assessing genomic instability. The relatively high frequency of MSI may also reflect peculiarities of this younger patient population. Occurrence of MSI or LOH was unrelated to clinical stage, nodal status, tumour size or grade or steroid receptor status. It was independent of mutations detected in exons 5-9 of the p53 gene. There was no significant association with survival. The lack of such correlations reflects a random disabling mechanism that may equally affect genes promoting cell death as well as growth.

Clinical implications of urokinase and tissue type plasminogen activators and their inhibitor (PAI-1) in breast cancer tissue.

Cytosol of primary breast cancers from 217 women of predominantly Arab ethnicity were assayed for uPA, tPA, PAI-1 and a subset for ER, PR and pS2. Serum levels of CEA and CA153 were determined during follow-up. Only tPA correlated to nodal status and tumour grade, and PAI-1 to clinical stage. PAI-1 was related to uPA and both were inversely correlated with PR and pS2 (PAI-1 also to ER). Conversely tPA was directly correlated with ER, PR and pS2. Women with high tumour uPA and PAI-1, but not tPA, had shorter overall, and relapse-free, survival. Only nodal status and clinical stage were independent predictors in multivariate analysis. However, uPA and PAI-1 were more prognostically informative than ER or PR and their usefulness may extend to delineation of patients likely to respond to adjuvant therapy.

Multifocal squamous cell carcinoma of the oesophagus following radiotherapy for bilateral breast carcinoma.

A 60 year old woman who presented with dysphagia and weight loss was found to have multiple foci of dysplasia and in situ and invasive squamous cell carcinoma scattered along the whole length of the oesophagus, with intervening areas of normal mucosa. The patient had a history of two breast carcinomas 19 and one year previously for which she had repeated radiotherapy. Several members of the patient's close family had histories of malignant disease. All oesophageal lesions and the more recent breast cancer showed positive immunostaining for p53 protein. p53 mutations, some involving different exons, were also detected in these lesions. No p53 immunostaining or mutations were detected in the normal oesophageal mucosa. The findings suggest an independent origin of the multiple dysplastic and neoplastic foci, which might have developed in a background of a field change, possibly related to the previous radiotherapy. The strong family history of malignant diseases raises the possibility that, in addition, genetic factors might have played a role in the development of the oesophageal disease.

Detection of Epstein-Barr virus in gastrectomy specimens.

Epstein Barr virus (EBV) has been reported to be present in a minority of gastric carcinomas and may be implicated in its pathogenesis. This study was aimed at determining the occurrence of EBV in 43 consecutive gastrectomy specimens with a variety of benign and malignant lesions. In situ hybridisation was used for detection of EBER RNA, the marker for latent EBV infection. Only 1/20 (5%) gastric cancers was EBER positive; a moderately differentiated adenocarcinoma with a heavy lymphocytic infiltration. The interstitial lymphocytic infiltrate was predominantly of B cell type, but the majority of lymphocytes overlying the tumour cells were CD8+ T cells. The other gastric lesions examined, which included 15 peptic ulcers, 6 stromal tumours and 2 lymphomas, were all EBER negative. Using a biotin detection system, scattered EBER positive cells were seen in adjacent normal gastric and/or duodenal mucosa in 9 sections from 8 cases (i.e., in 19% of all 43 cases examined). However, on using a digoxygenin detection system, no reactivity was found in these normal cells. An immunoperoxidase stain for chromogranin A showed that these apparently 'EBER positive' cells corresponded to normal chromogranin positive neuroendocrine cells within the gastric and duodenal mucosa. We conclude that EBV infection occurred only in the lymphoepithelioma type of gastric carcinoma and was absent from the other adenocarcinomas and from normal and benign tissues. The occasional EBER positive reaction encountered in normal cells was probably the result of a false signal arising from neuroendocrine cells as a consequence of the biotin-containing detection system.

Evaluation of prognostic factors in male breast cancer.

We conducted an analysis on 41 cases of male breast cancer (median age 54 y; range 25-82 y) in Kuwait. Most (51%) were stage II cancers with 65% arising in the left breast. There were 5 (12%) T1 tumours, 23 (56%) T2 tumours and 13 (32%) T3/T4 tumours. They were mostly (95%) infiltrating ductal carcinomas; 97% were grade 2 or 3. Axillary lymph node involvement was found in 69%. Estimated 5-year survival rates were 67% and 58% for overall and relapse free survival respectively. Favourable prognosis was associated with age below 50y, clinical stage I and II, small tumour size (T1, T2), low tumour grade and absence of nodal involvement or distant metastases; nodal status and grade were independent factors for relapse free survival in multivariate analysis. In 18 cases, an immunohistochemical study showed some degree of tumour antigen reaction for ER in 89% of cases, PR in 61%, pS2 in 44%, CathD in 72%, p53 in 56%, c-erbB-2 in 50%, Ki67 and PCNA in 100% and bcl-2 in 78%. There were significant associations between several of these factors but none influenced survival. Despite the high incidence of staining of ER, our data do not support the concept of an endocrine pathway that could be usefully antagonized with antioestrogens for therapeutic benefit, as in women.

Immunoradiometric measurement of pS2 in breast cancer--correlation with steroid receptors and plasminogen activators.

pS2 was measured by radioimmunometric assay in tumour extracts from 197 breast cancer patients. Values ranged from 0 to 50 ng/mg protein (mean 9.6 and median 3 ng/mg). We found no correlation with age, menopausal status, nodal metastases, disease stage or tumour histology. There was, however, a linear relationship with both ER (p < 0.0001) (particularly nuclear ER) and PR (p < 0.0001) expression determined by enzyme immunoassay (ELISA), as well as a good correlation when high and low expressors were stratified on the basis of combined ER/PR expression using consensus cut-off points. Only 15% of ER - ve/PR - ve patients were classified as pS2 + ve compared with 83% of those who were ER + ve/PR + ve. pS2 was also directly correlated with high expression of tPA and inversely with uPA. Comparison with previous studies showed that the current ELISA method produced consistent results, in contrast to other methods, particularly those based on immunohistochemical detection. The close relationship between pS2 and both steroid receptors suggests that pS2 may be important in terms of defining hormone-responsive patients who are likely to benefit from endocrine therapy.

Measurement of serum N-acetyl beta glucosaminidase activity in patients with breast cancer.

N-acetyl-beta-glucosaminidase (NAG) activity measured in sera from 129 breast cancer patients was elevated (mean 18.2 units/l) compared with that in sera from 28 healthy women (11.6 units/l) (p=0.001). There was a weak correlation between NAG activity and carcinoembryonic antigen (CEA) and CA-153, but no relationship to age, menopausal status, node status, stage, histology of tumour or to steroid receptors. NAG, CEA and CA-153 were measured in periodic follow-up samples taken after surgery (up to 26 months) from 17 patients. NAG activity fluctuated within a narrow range, unlike CEA and CA-153. In 70% of cases the pattern was similar to at least one of the other markers, and was generally maintained at a higher level in patients who suffered relapse compared with those who remained disease-free up to the last follow-up, but was not significantly altered before relapse. The measurement of NAG activity is unlikely to be of value in predicting time or occurrence of relapse or of clinical utility in post-surgical therapy. Increased appearance in serum may aid metastasis by degrading the extracellular matrix and it may be better investigated as a predictor of progression from in situ to invasive and metastatic cancer.

Modulation of elongation factor-1 delta (EF-1 delta) expression by oncogenes in human epithelial cells.

A cDNA clone covering part of the C-terminal domain of human EF-1 delta was isolated from mammary cancer cells by subtractive hybridisation. The higher expression of EF-1 delta in the tumours suggested that malignant transformation in vivo requires an increase in translation factor mRNA and protein synthesis for entry into and transition through the cell cycle. To explore the relation between cell division and EF-1 delta expression, MCF-7 cells were treated with dexamethasone, an inducer of differentiation. There was no change in the mRNA levels of EF-1 delta in the dexamethasone-treated cells. To explore the relation between oncogenes and EF-1 delta expression, a variety of oncogenes were introduced into human mammary epithelial cells (MCF-7) and human keratinocytes (HaCaT). Despite high oncogene mRNA expression, there was no significant change in the EF-1 delta mRNA level by v-src, c-erbB (EGF Receptor), c-erbB-2, v-myc and v-fos oncogenes. However, overexpression of v-Ha-ras in HaCaT cells resulted in a three to five-fold decrease in the steady-state mRNA level of EF-1 delta. Taken together, the data provides further support on the interaction of translation factors and oncogenic transformation.

CD5 positive breast carcinoma in a patient with untreated chronic lymphocytic leukaemia: molecular studies of chromosome 13q.

A 67 year old woman presented with a right breast lump which proved to be a grade 2 invasive ductal carcinoma with axillary lymph node metastasis. She had a five year history of CD5 positive chronic lymphocytic leukaemia, which never required treatment. Immunoperoxidase stains for CD5, using the monoclonal antibody NCL-CD-54C7, showed that there was extensive infiltration of axillary lymph nodes with CD5 positive B lymphocytes. Strong staining for CD5 was also seen in the carcinoma cells within the breast and lymph node metastases. It has recently been suggested that there is a tumour suppresser locus in chronic lymphocytic leukaemia at 13q12.3 near or at the BRCA2 locus. Deletion of regions on chromosome 13q containing the BRCA2 and RB1 genes has also been reported in sporadic breast cancers. These observations suggest that there may be a link between these two diseases acting through chromosome 13, but amplification of several microsatellite repeat markers failed to show any loss of heterozygosity or repeat instability at either these or several other loci on chromosome 13. Examination of additional such cases is needed to perform a more comprehensive study of the significance of positive CD5 staining of breast carcinoma.

Steroid receptor measurement in breast cancers: comparison between ligand binding and enzyme-immunoassay in cytosolic and nuclear extracts.

We have analysed cytoplasmic and nuclear extracts of breast-cancer tissue from a total of 799 patients, measuring both oestrogen and progesterone receptors (ER, PR) using either the ligand binding assay (LBA) or the enzyme immunoassay technique (EIA). Mean and median receptor levels were much lower than those widely reported by others. For ER, this may in part be a consequence of the younger median age of the patient group. The frequency of positivity, using consensus cut-off values for clinical evaluation, was also lower than that reported by the EORTC Receptor Study Group. Although the measurements comparing the 2 methods were statistically correlated in terms of positivity, based on the above criteria for clinical assessment, concordance was considered to be relatively poor, particularly for ER when assayed in the same samples by the 2 methods. In cytosolic but not nuclear extracts, the LBA method gave a higher median value for ER than the EIA (except in the group that had EIA values greater than 15 fmol/mg protein); for PR, median values were higher with EIA in both cell fractions. There was an excellent correlation between receptor amounts in cytosolic and nuclear extracts for both ER and PR using the EIA; this was significantly better than with LBA. We also observed a correlation between ER and PR in both cytosolic and nuclear fractions which was most pronounced when the analysis was done by EIA. The amounts of ER in the cytosolic fraction were also correlated with the those of PR in the nuclear fraction and ER in the nuclear fraction with PR in the cytosolic fraction, but only when the EIA method was used. We conclude that the EIA method appears to be more sensitive and gives biologically more reliable results. However, the disagreement between the methods may be due to legitimate recognition of altered forms of the receptor and may be of biological significance. Although the presence of receptor in the cytosolic fraction is artifactual, its measurement by EIA does parallel the amounts of nuclear receptor, which may be a more relevant biological parameter.

The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19.

The aim of this study was to determine whether reverse transcriptase polymerase chain reaction (RT-PCR) for keratin 19 (K19) provides additional information when combined with immunohistochemistry when used to detect micrometastases in blood and bone marrow in patients with primary breast cancer. We studied 78 patients with breast cancer who had no evidence of distant metastases. We collected blood and bone marrow, separated the mononuclear fraction and carried out RT-PCR and immunohistochemistry for K19. RT-PCR was done by two 40-cycle rounds using nested primers. In initial experiments, RT-PCR was shown to be capable of detecting one tumour cell in one million normal bone marrow cells, which was at least 10 times more sensitive than immunohistochemistry, while retaining specificity. Five per cent of the peripheral blood and 22% of the bone marrow samples contained K19 positive cells by immunohistochemistry staining. Using RT-PCR, these proportions increased to 25% and 35%, respectively. This represents a significantly greater detection frequency (P < 0.001 and P = 0.03, respectively). RT-PCR for K19 is a more sensitive method for detecting micrometastases in patients with primary breast cancer when compared with immunohistochemistry.

Expression of keratinocyte growth factor and its receptor in human breast cancer.

The level of expression of keratinocyte growth factor (KGF) mRNA has been measured in human breast cell lines, purified populations of epithelial cells, myoepithelial cells and fibroblasts from reduction mammoplasty tissue and a panel of 42 breast cancers and 30 non-malignant human breast tissues using a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) procedure. We found similar levels of KGF mRNA in malignant and non-malignant breast tissues. The study of the amount of KGF mRNA in breast cell lines and purified populations of cells revealed that fibroblasts are the predominant source of KGF with malignant and non-malignant epithelial cells containing very low levels of KGF mRNA. We have examined the distribution of fibroblast growth factor receptor (FGFR)-2-IIIb, which is a high-affinity receptor for KGF and find that it is present on malignant and non-malignant epithelial cells. The level of FGFR-2-IIIb present on breast cancer cell lines was sufficient for KGF stimulation of breast cancer cell proliferation. Other members of the fibroblast growth factor family have been either not expressed in the human breast (FGF3, FGF4) or have been found at much reduced levels in breast cancer (FGF1, FGF2) and this is the first member of the family to potentially influence the progression of breast cancer through stimulation of cell division.

Presence of exon 5-deleted oestrogen receptor in human breast cancer: functional analysis and clinical significance.

A variant form of the human oestrogen receptor (ER) mRNA lacking sequences encoded within exon 5 has been described (Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O'Malloy BW, McGuire WL 1991, Cancer Res 51: 105-109). We have examined the expression of the exon 5-deleted ER (HE delta5) mRNA variant in breast biopsies using reverse transcriptase polymerase chain reaction (RT - PCR). HE delta5 mRNA was present in only 13% of non-malignant breast tissues compared with 32% of carcinomas (95% CI, P=0.05). Presence of the HE delta5 mRNA was associated with the presence of immunohistochemically detected ER (P=0.015) and progesterone receptor (PR) (P=0.02). There was a positive correlation between the presence of HE delta5 and disease-free survival (P=0.05), suggesting that the presence of HE delta5 may be an indicator of better prognosis. We have raised a monoclonal antibody specific to the C-terminal amino acids of HE delta5. This antibody recognized the variant but not the wild-type ER protein. We show that HE delta5 protein is present in breast cancer using immunohistochemical techniques. We also analysed trans-activation by HE delta5 in mammalian cells and showed that, in MCF-7 cells, HE delta5 competes with wild-type ER to inhibit ERE-dependent trans-activation. Our results indicate that this variant is unlikely to be responsible for endocrine resistance of breast cancer, but its presence at both the mRNA and protein level suggest that it may, nevertheless, be involved in regulating the expression of oestrogen-responsive genes in breast cancer.