SERPINE1 and SMA expression at the invasive front predict extracapsular spread and survival in oral squamous cell carcinoma.

BACKGROUND: Extracapsular spread (ECS) in cervical lymph nodes is the single-most prognostic clinical variable in oral squamous cell carcinoma (OSCC), but diagnosis is possible only after histopathological examination. A promising biomarker in the primary tumour, alpha smooth muscle actin (SMA) has been shown to be highly prognostic, however, validated biomarkers to predict ECS prior to primary treatment are not yet available. METHODS: In 102 OSCC cases, conventional imaging was compared with pTNM staging. SERPINE1, identified from expression microarray of primary tumours as a potential biomarker for ECS, was validated through mRNA expression, and by immunohistochemistry (IHC) on a tissue microarray from the same cohort. Similarly, expression of SMA was also compared with its association with ECS and survival. Expression was analysed separately in the tumour centre and advancing front; and prognostic capability determined using Kaplan-Meier survival analysis. RESULTS: Immunohistochemistry indicated that both SERPINE1 and SMA expression at the tumour-advancing front were significantly associated with ECS (P<0.001). ECS was associated with expression of either or both proteins in all cases. SMA+/SERPINE1+ expression in combination was highly significantly associated with poor survival (P<0.001). MRI showed poor sensitivity for detection of nodal metastasis (56%) and ECS (7%). Both separately, and in combination, SERPINE1 and SMA were superior to MRI for the detection of ECS (sensitivity: SERPINE1: 95%; SMA: 82%; combination: 81%). CONCLUSION: A combination of SMA and SERPINE1 IHC offer potential as prognostic biomarkers in OSCC. Our findings suggest that biomarkers at the invasive front are likely to be necessary in prediction of ECS or in therapeutic stratification.

Nutlin-3, the small-molecule inhibitor of MDM2, promotes senescence and radiosensitises laryngeal carcinoma cells harbouring wild-type p53.

BACKGROUND: Primary radiotherapy (RT) is a mainstay of treatment for laryngeal squamous cell carcinoma (LSCC). Although the cure rates for early (T1) vocal cord tumours are high, RT proves ineffective in up to a third of T3 carcinomas. Moreover, RT is associated with debilitating early- and late-treatment-related toxicity, thus finding means to de-escalate therapy, while retaining/augmenting therapeutic effectiveness, is highly desirable. p53 is a key mediator of radiation responses; we therefore investigated whether Nutlin-3, a small-molecule inhibitor of MDM2 (mouse double minute 2; an essential negative regulator of p53), might radiosensitise LSCC cells. METHODS: We performed clonogenic assays to measure radiosensitivity in a panel of LSCC cell lines (for which we determined p53 mutational status) in the presence and absence of Nutlin-3. RESULTS: LSCC cells harbouring wild-type p53 were significantly radiosensitised by Nutlin-3 (P<0.0001; log-rank scale), and displayed increased cell cycle arrest and significantly increased senescence (P<0.001) in the absence of increased apoptosis; thus, our data suggest that senescence may mediate this increased radiosensitivity. CONCLUSION: This is the first study showing Nutlin-3 as an effective radiosensitiser in LSCC cells that retain wild-type p53. The clinical application of Nutlin-3 might improve local recurrence rates or allow treatment de-escalation in these patients.

Significance of the metastasis-inducing protein AGR2 for outcome in hormonally treated breast cancer patients.

The anterior gradient protein-2 (AGR2) is inducible by oestrogen and itself can induce metastasis in a rat model for breast cancer. Here, a rabbit antibody to recombinant human AGR2 was used to assess its prognostic significance in a retrospective cohort of 351 breast cancer patients treated by adjuvant hormonal therapy. The antibody stains 66% of breast carcinomas to varying degrees. The percentage of positive carcinoma cells in tumours directly correlates with the level of AGR2 mRNA (Spearman's rank correlation, P = 0.0007) and protein (linear regression analysis r2 = 0.95, P = 0.0002). There is a significant association of staining of carcinomas for AGR2 with oestrogen receptor alpha (ERalpha) staining and with low histological grade (both Fisher's Exact test P<0.0001). In the ERalpha-positive cases, but not the ERalpha-negative cases, when subdivided into the separate staining classes for AGR2, there is a significantly progressive decrease in patient survival with increased staining (log rank test, P = 0.006). The significant association of staining for AGR2 with patient death over a 10-year period (log rank test P = 0.007, hazard ratio = 3) only becomes significant at 6 years of follow-up. This may be due to the cessation of adjuvant hormonal therapy at an earlier time, resulting in adverse re-expression of the metastasis-inducing protein AGR2.

Correlation of mRNA for oestrogen receptor beta splice variants ERbeta1, ERbeta2/ERbetacx and ERbeta5 with outcome in endocrine-treated breast cancer.

This study has been performed to test the hypothesis that different oestrogen receptor beta (ERbeta) splice variants may be important determinants of clinical parameters, including outcome, in post-menopausal women with breast cancer receiving adjuvant endocrine treatment but no chemotherapy. Splice variants ERbeta1, ERbeta2 and ERbeta5 have been analysed by semi-quantitative RT-PCR in a cohort of 105 patients with primary breast cancer. Clinical correlates included age, grade, size, nodal status, ERalpha, progesterone receptor, Ki67, relapse-free survival (RFS) and overall survival (OS). Seventy per cent of cases were ERbeta1 positive, 69% ERbeta2 positive and 70% ERbeta5 positive. Within the cohort, 47% were positive for all three variants while 10% were negative for all three. ERbeta1 exhibited no discernible relationship with disease outcome. ERbeta2 and ERbeta5 expression was significantly associated with better RFS (P<0.005), and ERbeta2 with better OS (P=0.0002). In multivariate analysis, ERbeta2 (P=0.006), nodal status and the level of Ki67 expression were independent predictors for RFS while ERbeta2 (P=0.0008) and Ki67 status were independent predictors for OS. In the ERalpha-positive cases, or in the subset of those receiving adjuvant tamoxifen, ERbeta2 was significantly associated with good RFS (P<0.0005) and was the only independent marker of OS. We conclude that precise identification of splice variants of ERbeta are more important assessors than is ERbeta1 alone of the biological status of individual breast cancers, and hence in predicting their response to endocrine therapy.

Wild-type oestrogen receptor beta (ERbeta1) mRNA and protein expression in Tamoxifen-treated post-menopausal breast cancers.

This study has tested the hypothesis that comparison of protein and mRNA expression for ERalpha and ERbeta1 by human breast cancers provides novel information relating to the clinical and pathological characteristics of human breast cancers. Expression of ERalpha and ERbeta1 was identified in 167 invasive cancers from postmenopausal women treated only with endocrine therapy. The cohort included 143 cases receiving only adjuvant Tamoxifen following surgery. ERalpha and ERbeta1 expression was analysed by immunohistochemistry and reverse transcription RT-PCR and compared with clinical progression of individual cancers. ERalpha protein was closely associated with the corresponding RNA detected by RT-PCR (Chi-square, P<0.001). In contrast, ERbeta1 protein and mRNA were inconsistent. Although an association was identified between ERalpha and ERbeta mRNAs (Chi-square, P<0.001) and between ERalpha protein and ERbeta1 mRNA (Chi-square, P<0.027), no association was identified for the ERalpha and ERbeta1 proteins detected by immunohistochemistry. ERbeta1 was not associated with outcome. However, in the absence of ERalpha, ERbeta1 protein expression was associated with elevated cell proliferation. There was a trend for the ERbeta1 protein-positive cases to have a worse outcome, both within the group as a whole as well as within the ERalpha-positive Tamoxifen-treated cases. This study has confirmed the hypothesis that expression of ERalpha is an important determinant of breast cancer progression, and has further demonstrated that ERbeta1 may play a role in the response of breast cancers to endocrine therapy.

Increased risk of malignant progression in benign proliferating breast lesions defined by expression of heat shock protein 27.

Heat shock protein 27 (hsp-27) is a regulator of oestrogen receptor (ER) expression and a modulator of intracellular homeostasis. In this laboratory, Shaaban et al demonstrated the importance of ER-alpha, together with Ki67, in enhancing the progression of benign breast lesions of defined morphological types. To better understand the mechanisms by which ER-alpha promotes breast neoplasia, this study was performed to test the hypothesis that the roles of ER-alpha and hsp-27 may be defined by their quantitative expression in proliferative breast lesions of varying histological risk. The expression of hsp-27 was identified using a specific monoclonal antibody and analysed to assess the proportion of positive epithelial cells using digitised morphometric image analysis. The expression of ER-alpha was analysed by immunohistochemistry and Western blotting in a variety of benign (HUMA121) and malignant mammary cell lines, including ER-alpha(+) (MCF7, ZR-75, T47D) and ER-alpha(-) (MDA-MB 231) breast cancer cell lines. The data confirm that, during progression from normal through proliferative breast lesions to in situ cancer, there was a significant increase in both the proportion and the optical density of the epithelial cells expressing hsp-27. The mean levels of expression ranged from 7.4% of the total number of epithelial cells in normal lobules to 25.17% of epithelial cells in hyperplasias of usual type (HUT) to 61.1% of epithelial cells in ductal carcinoma in situ (P<0.001). The study has confirmed the expression of hsp-27 to be closely associated with ER-alpha(+) expression, and that its regulated expression occurs early along the mammary oncogenic pathway, supporting the initial hypothesis. It is our proposal that the differential expression of hsp-27 modulates the phenotypic behaviour of morphologically benign epithelial cells and hence may be an important determinant in initiating, or promoting, a population of human mammary cancers.

Identification of mRNAs differentially-expressed between benign and malignant breast tumour cells.

Two suppression subtracted cDNA libraries have been constructed, one containing cDNAs to mRNAs present at a higher level in a benign human breast tumour-derived cell line relative to the malignant mammary cell line, MCF-7, and the other containing cDNAs present at a higher level in the MCF-7 cells relative to the benign cells. Randomly-picked cloned DNAs have been sequenced yielding 29 and 128 different cDNAs from the benign and malignant libraries, respectively. Using reverse Northern hybridisation, 76% and 83% of the cDNAs were differentially expressed by greater than two-fold, whilst 14% and 11% of cDNAs in the respective libraries were differentially expressed by more than 15-fold. Amongst these were oestrogen-responsive cDNAs and expressed sequence tags. One such oestrogen-responsive expressed sequence tag, M41, is transcribed from a gene located on chromosome 21q22.3, within an intron of a larger gene. The M41 gene contains oestrogen response elements, one of which is associated with alu repeats. M41 mRNA is expressed at a statistically significantly higher level in human breast cancer specimens than in normal human breast and benign lesions. In carcinomas, its up-regulation is associated with the development of the malignant cell.

Molecular indexing of human genomic DNA.

Molecular indexing sorts DNA fragments into subsets for inter-sample comparisons. Type IIS or interrupted palindrome restriction endonucleases, which result in single-stranded ends not including the original recognition sequence of the enzyme, are used to produce the fragments. The ends can then be any sequence but will always be specific for a given fragment. Fragments with particular ends are selected by ligation to a corresponding indexing adapter. We describe iterative indexing, a new process that after an initial round of indexing uses a Type IIS restriction endonuclease to expose additional sequence for further indexing. New plasmids, pINDnn, were produced for novel use as indexing adapters. Together, the plasmids index all 16 possible dinucleotides. Their large size can be increased by dimerisation in vitro and allows the isolation of indexed material by size separation. Fragments produced from human genomic DNA by Type II restriction endonucleases were sorted using six bases in total to a possible enrichment of 1920-fold. By comparison with the public human sequence databases, fidelity of indexing was shown to be high and was tolerant of repetitive sequences. Genome-wide comparisons on a candidate or non-candidate basis are made possible by this approach.

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA.

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.

Characterisation of molecular alterations in microdissected archival gliomas.

Classification of gliomas according to their molecular characteristics may be important in future histopathological diagnosis. However, gliomas frequently display heterogeneity at the histological, biological and molecular level. In this study of archival diagnostic gliomas, precision microdissection was used to enrich samples in the most malignant cells or to investigate intratumoural histological heterogeneity. Analysis of tumour samples microdissected from the most aggressive regions, representative of the histopathological diagnosis, revealed PTEN mutations in 4/14 anaplastic astrocytomas, 4/13 glioblastomas and 1 gliosarcoma, but not in 19 low-grade gliomas. Using a novel PCR procedure and direct sequence analysis of the entire coding sequence, TP53 mutations were detected in 1/3 pilocytic astrocytomas, 3/13 astrocytomas, 4/14 anaplastic astrocytomas, 5/13 glioblastomas and 1 gliosarcoma. All but one of the tumours with TP53 mutation showed p53 immunopositivity, but 5 low-grade and 10 high-grade gliomas had p53 protein nuclear accumulation in the absence of detectable mutation. p53 status was unrelated to p21 expression. Neither PTEN nor TP53 mutations influenced the proliferative index or microvessel density of high-grade astrocytomas. Unusual findings include: TP53 mutation in a juvenile pilocytic astrocytoma; TP53 and PTEN mutations in a de novo glioblastoma, a gliosarcoma with identical mutations in gliomatous and sarcomatous components, and an infratentorial anaplastic astrocytoma with an earlier supratentorial grade II astrocytoma bearing the same TP53 mutation but not the PTEN mutation or loss of heterozygosity (LOH) of 10q23. Similarly, the transition to high-grade histology was associated with acquisition of PTEN mutations and 10q23.3 LOH in two de novo high-grade tumours with regions of low-grade histology.

Immunodetectable cyclin D(1)is associated with oestrogen receptor but not Ki67 in normal, cancerous and precancerous breast lesions.

Cyclin D1 is associated with cell cycle regulation and has more recently been shown to stimulate the transcriptional functions of the oestrogen receptor (ER). Furthermore, in normal breast there is a negative association between expression of ER and the proliferation marker Ki67 indicating that either ER positive cells are non-dividing or that the receptor is down-regulated as cells enter cycle. This important relationship breaks down in many ER-positive cancers and precancerous breast lesions where the receptor is often detected on proliferating cells. The aims of the present study were to determine the interplay between ER, Ki67 and cyclin D(1)in individual cells within the spectrum of human breast lesions ranging from normal to invasive carcinoma by using dual staining immunofluorescence. We found that in normal breast there was a strong positive association between ER and cyclin D(1)expression. In contrast there was a strong negative association between cyclin D(1)and Ki67 expression. Similar findings were seen for the other precancerous and cancerous breast lesions. Thus immunodetectable cyclin D(1)within individual cells does not appear to be associated with cell cycle progression in the benign or malignant breast but instead may have important interactions with ER.

Subgroups of non-atypical hyperplasia of breast defined by proliferation of oestrogen receptor-positive cells.

In normal breast, there is a negative association between expression of oestrogen receptor (ER) and the proliferation marker Ki67, indicating that ER-positive (ER+) cells do not divide, or that the receptor is down-regulated when they do so. However, dual staining has been found in carcinomas and precancerous lesions, indicating that abnormal regulation of ER could be important in breast tumourigenesis. ER expression in relationship to cell proliferation was studied in 241 foci of hyperplasia of usual type (HUT), a lesion associated with a 1.5 to 2-fold increase in risk of developing breast cancer. Dual label immunofluorescence was employed, using the antibodies 1D5 and Ki67. Two hundred and thirteen foci of HUT contained ER+ cells, which were distributed singly or contiguously and increased with age. Most foci resembled normal breast, but 51 contained dual labelled cells, which did not increase with age. Some of these foci exhibited few, scattered ER+ cells with greater proliferation rates than the ER-negative (ER-) cells, whereas others contained many, contiguous ER+ cells, whose rate of proliferation was less than that of the ER- cells. The latter picture is similar to that which has previously been reported in atypical ductal hyperplasia and ductal carcinoma in situ. The first type of HUT may evolve into the second. The proportion of Ki67+ cells that was ER+ was similar in both types, suggesting a homeostatic mechanism that slows the proliferation of ER+ cells as they become confluent. Overriding this inhibition may be crucial in further progression. Non-atypical hyperplasia is thus heterogeneous in ER expression and proliferation and a significant proportion exhibit abnormal regulation of ER. These findings could have implications for pathological diagnosis, risk assessment, and prophylactic hormonal therapy.

Abnormal regulation of the oestrogen receptor in benign breast lesions.

BACKGROUND: In normal breast tissue the oestrogen receptor (ER) and the proliferation associated antigen Ki67 are negatively associated, indicating that ER+ cells are non-dividing, or that the receptor is downregulated as cells enter cycle. This relation is completely or partially lost in many ER+ breast cancers and in in situ proliferations associated with an increased cancer risk, where coexpression of the two markers is often found. AIMS: To determine whether similar changes can be identified in other risk associated breast lesions. PATIENTS/METHODS: Paraffin wax blocks from 12 cases of lactational change, 21 apocrine metaplasias, 22 duct ectasias, 20 sclerosing adenosis, 20 fibroadenomas, 19 phyllodes tumours, 20 radial scars, 21 papillomas (15 solitary and six multiple), 15 gynaecomastias, and nine postmortem male breast tissues were retrieved. Immunohistochemistry was used to determine the expression of ER and dual labelling immunofluorescence was used to detect cells expressing both ER and Ki67. RESULTS: Increased numbers of ER+ cells were seen in sclerosing adenosis, radial scars, papillomas, fibroadenomas, and phyllodes tumours but not in apocrine cysts (where no ER+ cells were detected) or duct ectasia (where normal numbers were found). As in the normal breast, the proportion of ER+ cells increased with age in all lesions with the exception of fibroadenomas. Coexpression of ER and Ki67 was found in an increased proportion of cells of all risk associated lesions studied. ER+ cells were less likely to be dividing than ER- cells in all cases, although this was significant only for sclerosing adenosis. The data on sclerosing adenosis, radial scars, papillomas, and fibroadenomas are comparable with those reported previously in hyperplasia of usual type, whereas those in duct ectasia are similar to those of the normal breast. The findings in all lesions, however, differed from those in ductal carcinoma in situ, where proportions of ER+ and ER+/Ki67+ cells are higher and the relation between ER+ cell numbers and age is lost. Thus, the nature and degree of dysregulation of ER in benign breast lesions is broadly in accordance with the degree of risk of developing breast cancer with which they are associated. In gynaecomastia, the proportions of ER+ and ER+/Ki67+ cells were comparable with those seen in benign female breast lesions, but changes with age were not observed. However, the changes in gynaecomastia were similar to those seen in normal male breast. CONCLUSION: These findings are in keeping with the contention that the dissociation of ER and Ki67 expression is a very early change in the pathway to many breast cancers. However, this change might only have preneoplastic importance in the hormonal milieu of the female breast.

Expression of calcium-binding protein S100A2 in breast lesions.

A suppression subtraction cDNA library representing mRNAs expressed at a higher level in a benign breast tumour-derived cell line relative to the malignant MCF-7A cell line contained cDNAs corresponding to mRNAs for plasminogen activator inhibitor I, annexin VIII and the EF-hand protein S100A2. S100A2 protein has previously been shown to be expressed in normal human breast epithelium, but not in human breast carcinoma cell lines. Using a PCR-based assay and in situ hybridization on histological sections of human breast specimens, the mRNA for S100A2 was shown to be present in all benign breast lesions examined as well as in normal epithelium. S100A2 mRNA was detectable in 37% of specimens of carcinoma in situ, but in less than 15% of carcinoma specimens. The results suggest that the loss of S100A2 is associated with the development of malignant cells and is not associated with early tumour development.

Molecular genetic analysis of archival gliomas using diagnostic smears.

Investigation of the clinical significance of genetic alterations in gliomas requires molecular genetic analysis using samples from retrospective or prospective clinical studies. However, diagnostic tissue is often severely limited and because of fixation, paraffin-embedded tissues (PET) contain degraded DNA. Intra-operative cytological preparations (smears) archived after diagnosis may represent an additional source of clinical material for genetic analysis. In this study, tissue samples were obtained by precision microdissection of archived diagnostic smears from 20 cases (1961-1999). All samples produced polymerase chain reaction (PCR) products for the beta globin gene, but the most recent samples amplified best and gave longer amplimers. For six cases, direct comparison was made between samples microdissected from smears and the corresponding PET. Samples from smears showed improved PCR performance and similar alleles on microsatellite marker analysis. One case, with smears of uninvolved cortex and tumour tissue available for microdissection, showed allelic imbalance at 10q23 on the basis of the smear results alone. PCR products from smears were shown to be suitable for direct sequence analysis (p53 gene). A PTEN mutation, found previously in an anaplastic astrocytoma by analysis of PET, was detected in the corresponding diagnostic smear. The results of this study indicate that tissue samples microdissected from diagnostic intra-operative cytological preparations may be suitable for molecular genetic analysis of gliomas.

Novel polymerase chain reaction approach for full-coding p53 mutation detection in microdissected archival tumors.

Evidence suggests that up to 25% of p53 mutations are outside of exons 5-8 and that insertions, deletions, and polymorphic sites in the p53 gene may play a significant role in the process of carcinogenesis. A novel polymerase chain reaction (PCR) approach for the analysis of the entire p53 coding and splice site regions from microdissected, formalin-fixed, paraffin-embedded tumor tissues has been developed which allows multiple genetic analyses to be performed from one primary amplification reaction. The method was initially evaluated using well-characterized cell lines. In addition to confirming the published p53 mutations for HT29, Molt 4, A431, and HN5, a 16 base pair (bp) duplication within intron 3 was detected in both the A431 and HT29 cell lines. Analysis of archival samples of ovarian cancer identified the same 16-bp duplication and coding region variations. In all samples, using GenBank submission U94788 as a reference, a C-insertion was detected at nucleotide positions 11818 and 11874 within intron 2. At nucleotide position 14168, within intron 7, a T-to-G base change was found. This novel PCR approach has the potential to reduce the amount of clinical material required by up to 95%, thus facilitating retrospective studies on archival tumor collections. Furthermore, a wider analysis of the p53 gene, including splice sites and intronic regions, may yield additional information regarding cancer predisposition, response to therapy, and progression.

Estrogen receptor-positive proliferating cells in the normal and precancerous breast.

Recently it has been shown that epithelial cell expression of the estrogen receptor (ER) and that of the proliferation-associated marker Ki-67 are almost mutually exclusive in the normal premenopausal human breast but that coexpression frequently occurs in estrogen receptor-positive (ER+) breast cancers. This coexpression may indicate disordered expression of ER in the cell cycle or failure to suppress division of ER+ cells and could be important in neoplastic transformation. The purpose of this study was to determine whether in situ proliferations known to be associated with different levels of risk for developing breast cancer contain these coexpressing cells and, if so, the stage at which they occur. We found that ER+ proliferating cells were rare in premenopausal lobules but increased with age in the normal breast. There was no difference in nonlesional tissue between cancerous and noncancerous breasts. The percentage of dual-expressing cells was significantly increased, however, in all of the in situ proliferations and correlated positively with the level of risk of developing breast cancer. We suggest that development of at least some human breast cancers is associated with increasing failure to down-regulate ER as cells enter the cycle or to suppress division of ER+ cells. The mechanism may involve the loss of a tumor suppressor gene.

Oestrogen receptor expression in the normal and pre-cancerous breast.

As oestrogen is associated with most of the epidemiological risk factors for breast cancer, the number and distribution of oestrogen receptor positive (ER+) cells could have a bearing on the development of the disease. ER+ cells were thus studied in the normal breast and in the spectrum of in situ proliferations which range from non-atypical hyperplasia to in situ carcinoma and are associated with different levels of risk for developing breast cancer. In the normal pre-menopausal breast, ER+ cells comprised the minority and were distributed singly, being surrounded by oestrogen receptor negative (ER-) cells. ER+ cells showed a statistically significant increase with age, reaching a plateau after the menopause, and the increase was associated with a tendency for positive cells to become contiguous in patches of variable size. A small proportion of lobules showing involutional change comprised over 90 per cent ER+ cells. The significance of this feature is not clear but no evidence was found that it was pre-cancerous. The percentage of ER+ cells was slightly increased in hyperplasia of usual type (non-atypical hyperplasia, HUT) and the relationship to age was maintained. The staining pattern was variable; in some lesions ER+ cells were surrounded by ER- cells whereas in others there were contiguous groups of positive cells sometimes accounting for more than 90 per cent of cells in the lesion. In contrast, all cases of atypical ductal hyperplasia (ADH), lobular in situ neoplasia (LIN) and ductal carcinoma in situ (DCIS) exhibited positivity of contiguous cells accounting for the majority in the lesions. Furthermore, the relationship between ER+ cell numbers and age was lost in these lesions, indicating autonomy of ER expression or of proliferation of cells expressing the receptor. It is hypothesized that this dysregulation of receptor expression or of ER+ cell numbers at the ADH stage may be the precursor of abnormal expression of cyclins and other cell cycle control proteins which have been shown first to appear in DCIS.

Mutation in the PTEN/MMAC1 gene in archival low grade and high grade gliomas.

The PTEN gene, located on 10q23.3, has recently been described as a candidate tumour suppressor gene that may be important in the development of advanced cancers, including gliomas. We have investigated mutation in the PTEN gene by direct sequence analysis of PCR products amplified from samples microdissected from 19 low grade (WHO Grade I and II) and 27 high grade (WHO grade III and IV) archival, formalin-fixed, paraffin-embedded gliomas. Eleven genetic variants in ten tumours have been identified. Eight of these are DNA sequence changes that could affect the encoded protein and were present in 0/2 pilocytic astrocytomas, 0/2 oligoastrocytomas, 0/1 oligodendroglioma, 0/14 astrocytomas, 3/13 (23%) anaplastic astrocytomas and 5/14 (36%) glioblastomas. PTEN mutations were found exclusively in high grade gliomas; this finding was statistically significant. Only two of the PTEN genetic variants have been reported in other studies; two of the genetic changes are in codons in which mutations have not been found previously. The results of this study indicate that mutation in the PTEN gene is present only in histologically more aggressive gliomas, may be associated with the transition from low histological grade to anaplasia, but is absent from the majority of high grade gliomas.