D R Sibson

Wild-type oestrogen receptor beta (ERβ1) 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 ERα and ERβ1 by human breast cancers provides novel information relating to the clinical and pathological characteristics of human breast cancers. Expression of ERα and ERβ1 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. ERα and ERβ1 expression was analysed by immunohistochemistry and reverse transcription RT–PCR and compared with clinical progression of individual cancers. ERα protein was closely associated with the corresponding RNA detected by RT–PCR (Chi-square, P<0.001). In contrast, ERβ1 protein and mRNA were inconsistent. Although an association was identified between ERα and ERβ mRNAs (Chi-square, P<0.001) and between ERα protein and ERβ1 mRNA (Chi-square, P<0.027), no association was identified for the ERα and ERβ1 proteins detected by immunohistochemistry. ERβ1 was not associated with outcome. However, in the absence of ERα, ERβ1 protein expression was associated with elevated cell proliferation. There was a trend for the ERβ1 protein-positive cases to have a worse outcome, both within the group as a whole as well as within the ERα-positive Tamoxifen-treated cases. This study has confirmed the hypothesis that expression of ERα is an important determinant of breast cancer progression, and has further demonstrated that ERβ1 may play a role in the response of breast cancers to endocrine therapy.

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 (Spearmans 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 α (ERα) staining and with low histological grade (both Fishers Exact test P<0.0001). In the ERα-positive cases, but not the ERα-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.

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.

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 D1in 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 D1expression. In contrast there was a strong negative association between cyclin D1and Ki67 expression. Similar findings were seen for the other precancerous and cancerous breast lesions. Thus immunodetectable cyclin D1within 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.

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

SummaryThe 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.

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-α, together with Ki67, in enhancing the progression of benign breast lesions of defined morphological types. To better understand the mechanisms by which ER-α promotes breast neoplasia, this study was performed to test the hypothesis that the roles of ER-α 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-α was analysed by immunohistochemistry and Western blotting in a variety of benign (HUMA121) and malignant mammary cell lines, including ER-α(+) (MCF7, ZR-75, T47D) and ER-α(−) (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-α(+) 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.

Characterisation of molecular alterations in microdissected archival gliomas.

Abstract. 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.

CYP2B6*6 is an independent determinant of inferior response to fludarabine plus cyclophosphamide in chronic lymphocytic leukemia

Fludarabine plus cyclophosphamide (FC) is the chemotherapy backbone of modern chronic lymphocytic leukemia (CLL) treatment. CYP2B6 is a polymorphic cytochrome P450 isoform that converts cyclophosphamide to its active form. This study investigated the possible impact of genetic variation in CYP2B6 on response to FC chemotherapy in CLL. Available DNA samples from the LRF CLL4 trial, which compared chlorambucil, fludarabine, and FC, were screened by TaqMan real-time polymerase chain reaction assays for CYP2B6 SNPs c.516G>T and c.785A>G, which define the most common variant allele (*6). Among the 455 samples successfully genotyped, 265 (58.2%), 134 (29.5%), and 29 (6.4%) were classified as *1/*1, *1/*6, and *6/*6, respectively. Patients expressing at least one *6 allele were significantly less likely to achieve a complete response (CR) after FC (odds ratio 0.27; P = .004) but not chlorambucil or fludarabine. Analysis of individual response indicators confirmed that this inferior response resulted from impaired cytoreduction rather than delayed hemopoietic recovery. Multivariate analysis controlling for age, gender, stage, IGHV mutational status, 11q deletion, and TP53 deletion/mutation identified CYP2B6*6 and TP53 mutation/deletion as the only independent determinants of CR attainment after FC. Our study provides the first demonstration that host pharmacogenetics can influence therapeutic response in CLL. This trial is registered as an International Standard Randomised Control Trial, number NCT 58585610 at www.clinicaltrials.gov.

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 β 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.

Abstract 5246: Genomic determinants of aggressive phenotype in oral SCC

Background: Oral squamous cell carcinoma (OSCC), the common form of HNSCC, affects over 600,000 cases worldwide per annum. We reviewed 561 consecutive cases in Liverpool 1 finding that the most predictive marker for death was extra-capsular spread (ECS) from cervical lymph nodes (p 2 . The presence of ECS predicted for recurrence, usually at the primary site, even when surgical margins were generous indicating that this aggressive phenotype is likely to be expressed in the primary tumour as much as in the metastatic cells. Hypothesis: We hypothesised that molecular pathways represented by global gene expression signatures would be detected in the samples associated with ECS. Methods: High density exon array expression analysis was performed on fresh frozen samples comprising T2 and T4 stage OSCC cases having no nodal involvement; positive nodes without ECS or positive nodes plus ECS picked longitudinally from a single centre series (n>200) with bias towards increasing the numbers with ECS. Contingency tests were performed to assess the significance of ECS associated pathways. Results and Discussion: Power analysis showed that 90% of all ≥ twofold differences in gene expression between node negative & ECS cases were significant. Affected pathways included: keratinocyte differentiation, ECM remodelling, chemokines & adhesion, TGF & WNT: cytoskeletal remodelling, regulation of actin cytoskeleton by rho GTPases & cell adhesion/ histamine H1 receptor signalling/loss of cell barrier (p values:7.7×10 −15 – 1.9×10 −6 ). A 29 gene signature able to discriminate the ECS from the non-ECS cases with 78% sensitivity and 86% specificity was found. Blocked differentiation of keratinocytes at an early (“stem cell”/ EMT) stage was suggested by the genes which included keratin 15, keratinocyte differentiation-associated protein, retinol binding protein 1, cellular serine peptidase inhibitor (Kazal type 6), and stratifin implicated in mesenchymal stem cells, basal, suprabasal expression and keratinocyte activation and differentiation. The signature may be connected with these tumours having poor prognosis. Conclusion: This is a significant and novel finding suggesting that the pattern of metastasis in regional lymph nodes can be predicted by molecular pathways prevalent in a small oral biopsy. 1. Rogers et al. Oral Oncol 2009;45(3):201-211 2. Shaw et.al. Head Neck 2010;32(6):714-722 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5246. doi:10.1158/1538-7445.AM2011-5246