Sandra Stein

Methylome sequencing in triple-negative breast cancer reveals distinct methylation clusters with prognostic value

Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, our findings demonstrate the feasibility of profiling the cancer methylome with limited archival tissue to identify regulatory regions associated with cancer.

Bright-field in situ hybridization for HER2 gene amplification in breast cancer using tissue microarrays: correlation between chromogenic (CISH) and automated silver-enhanced (SISH) methods with patient outcome.

HER2 gene amplification or overexpression occurs in 15% to 25% of breast cancers and has implications for treatment and prognosis. The most commonly used methods for HER2 testing are fluorescence in situ hybridization (FISH) and immunohistochemistry. FISH is considered to be the reference standard and more accurately predicts response to trastuzumab, but is technically demanding, expensive, and requires specialized equipment. In situ hybridization is required to be eligible for adjuvant treatment with trastuzumab in Australia. Bright-field in situ hybridization is an alternative to FISH and uses a combination of in situ methodology and a peroxidase-mediated chromogenic substrate such as diaminobenzidine [chromogenic in situ hybridization (CISH)] or multimer technology coupled with enzyme metallography [silver-enhanced in situ hybridization (SISH)] to create a marker visible under bright-field microscopy. CISH was introduced into diagnostic testing in Australia in October 2006. SISH methodology is a more recent introduction into the testing repertoire. An evaluation of CISH and SISH performance to assess patient outcome were performed using tissue microarrays. Tissue microarrays were constructed in duplicate using material from 593 patients with invasive breast carcinoma and assessed using CISH and SISH. Gene amplification was assessed using the American Society of Clinical Oncology/College of American Pathologists guideline and Australian HER2 Advisory Board criteria (single probe: diploid, 1 to 2.5 copies/nucleus; polysomy >2.5 to 4 copies/nucleus; equivocal, >4 to 6 copies/nucleus; low-level amplification, >6 to 10 copies/nucleus and high-level amplification >10 copies/nucleus; dual probe HER2/CHR17 ratio: nonamplified 2.2). Results were informative for 337 tissue cores comprising 230 patient samples. Concordance rates were 96% for HER2 single probe CISH and SISH and 95.5% for single probe CISH and dual probe HER2/CHR17 SISH. Both bright-field methods correlated with immunohistochemistry results and with breast cancer-specific survival. HER2 SISH testing combines the advantages of automation and bright-field microscopy to facilitate workflow within the laboratory, improves turnaround time, and correlates with patient outcome.Introduction HER2 gene amplification or overexpression occurs in 15% to 25% of breast cancers and has implications for treatment and prognosis. The most commonly used methods for HER2 testing are fluorescence in situ hybridization (FISH) and immunohistochemistry. FISH is considered to be the reference standard and more accurately predicts response to trastuzumab, but is technically demanding, expensive, and requires specialized equipment. In situ hybridization is required to be eligible for adjuvant treatment with trastuzumab in Australia. Bright-field in situ hybridization is an alternative to FISH and uses a combination of in situ methodology and a peroxidase-mediated chromogenic substrate such as diaminobenzidine [chromogenic in situ hybridization (CISH)] or multimer technology coupled with enzyme metallography [silver-enhanced in situ hybridization (SISH)] to create a marker visible under bright-field microscopy. CISH was introduced into diagnostic testing in Australia in October 2006. SISH methodology is a more recent introduction into the testing repertoire. An evaluation of CISH and SISH performance to assess patient outcome were performed using tissue microarrays. Materials and Methods Tissue microarrays were constructed in duplicate using material from 593 patients with invasive breast carcinoma and assessed using CISH and SISH. Gene amplification was assessed using the American Society of Clinical Oncology/College of American Pathologists guideline and Australian HER2 Advisory Board criteria (single probe: diploid, 1 to 2.5 copies/nucleus; polysomy >2.5 to 4 copies/nucleus; equivocal, >4 to 6 copies/nucleus; low-level amplification, >6 to 10 copies/nucleus and high-level amplification >10 copies/nucleus; dual probe HER2/CHR17 ratio: nonamplified <1.8, equivocal 1.8 to 2.2, amplified >2.2). Results Results were informative for 337 tissue cores comprising 230 patient samples. Concordance rates were 96% for HER2 single probe CISH and SISH and 95.5% for single probe CISH and dual probe HER2/CHR17 SISH. Both bright-field methods correlated with immunohistochemistry results and with breast cancer-specific survival. Conclusions HER2 SISH testing combines the advantages of automation and bright-field microscopy to facilitate workflow within the laboratory, improves turnaround time, and correlates with patient outcome.

Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer

MicroRNAs (miRNAs) are small non-coding RNAs of ∼20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located ∼2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.

Circulating Cell-Free Tumour DNA in the Management of Cancer

With the development of new sensitive molecular techniques, circulating cell-free tumour DNA containing mutations can be identified in the plasma of cancer patients. The applications of this technology may result in significant changes to the care and management of cancer patients. Whilst, currently, these “liquid biopsies” are used to supplement the histological diagnosis of cancer and metastatic disease, in the future these assays may replace the need for invasive procedures. Applications include the monitoring of tumour burden, the monitoring of minimal residual disease, monitoring of tumour heterogeneity, monitoring of molecular resistance and early diagnosis of tumours and metastatic disease.

Evaluation of oestrogen and progesterone receptor status in HER‐2 positive breast carcinomas and correlation with outcome

Aim: HER‐2/neu amplification occurs in 15–25% of breast carcinomas. This oncogene, also referred to as c‐erbB‐2, encodes a transmembrane tyrosine kinase receptor belonging to the epidermal growth factor receptor family. HER‐2 over‐expression is reported to be associated with a poor prognosis in breast carcinoma patients and in some studies is associated with a poorer response to anti‐oestrogen therapy. These patients are less likely to benefit from CMF (cyclophosphamide, methotrexate, fluorouracil)‐based chemotherapy compared with anthracycline‐based chemotherapy. The aim of this study was to evaluate breast carcinomas to determine hormone receptor status and if there is a difference in breast cancer specific survival for HER‐2 positive patients. Methods: A total of 591 breast carcinomas were evaluated using immunohistochemistry (IHC) for oestrogen receptor (ERp), progesterone receptor (PRp) and three different HER‐2 antibodies (CB11, A0485 and TAB250). Percentage of tumour cells and intensity of staining for ERp were evaluated using a semiquantitative method. Results: Of the 591 tumours, 91 (15.4%) showed 3+ membrane staining for HER‐2 with one or more antibodies. Of these 91 tumours, 41 (45.1%) were ERp+/PRp+, seven (7.7%) were ERp+/PR–, six (6.6%) were ERp–/PRp+ and 37 (40.7%) were ERp–/PR–. Of HER‐2 positive tumours, 5.5% showed >80% 3+ staining for ERp compared with 31.8% of 0–2+ HER‐2 tumours; 24.2% of HER‐2‐positive tumours showed 60% or more cells with 2+ or 3+ staining for ERp. Treatment data were available for 209 patients and no difference was observed in breast cancer specific survival (BCSS) with HER‐2 status and tamoxifen. Conclusion: Oestrogen receptor status cannot be used to select tumours for evaluation of HER‐2 status, and oestrogen and progesterone receptor positivity does not preclude a positive HER‐2 status. There is a higher proportion of ERp negative tumours associated with HER‐2 positivity, however, more than 20% of HER‐2 positive tumours show moderate or strong staining for ERp. HER‐2 positive patients in this study did not show an adverse BCSS with tamoxifen treatment unlike some previous studies.

Prediction of histologic grade in breast cancer using an artificial neural network

Histological grade is a historically used and well-documented prognostic indicator in breast cancer. There are three categories of grade (G1, G2 and G3) based on the degree of tubule formation, nuclear pleomorphism and mitotic count. A number of studies have reported that histological assessment is not uniformly reported. As a result of low inter-pathologist correlation associated with pathological diagnosis and non-standardised grading systems, patients are not always allocated into the correct grouping, and G2 has often been considered a “safe” group if one is unsure. A previously published study used real-time polymerase chain reaction (RT-PCR) for 5 genes to molecularly classify the G2 tumours into either G1 or G3. Due to the workflow constraints within pathology laboratories it was not considered feasible to molecularly profile every G2 tumour. In light of this we obtained the antibodies that corresponded to the 5 genes (BUB1B, CENPA, RACGAP1, RRM2 and NEK2) and performed immunohistochemistry (IHC) on formalin fixed paraffin embedded (FFPE) sections of 43 tumours (11 G1 and 32 G3). Results for all tumours were randomly divided into training and testing sets and an artificial neural network (NeuralSight and NeuralWare Predict) was used to classify the grade of tumours. Thirty-three additional G2 tumours were used for validation of the ANN. The ANN classified these tumours into 5 G1 and 28 G3 tumours. This predicted grade showed significant correlation with patient survival. Neural networks can be used to reclassify breast cancer G2 tumours into G1 and G3 using a panel of 5 IHC markers. This has the potential to impact on patient care, treatment decisions and outcome.

Morphological and molecular analysis of a breast cancer cluster at the ABC Studio in Toowong

Sir, Cancer clusters involving small numbers of patients are relatively common, but only a few have been investigated. In the case of breast cancer, for some putative clusters no significant increase in the risk of developing cancer has been found, and in only a minority of cancer clusters which are found to be statistically significant is a causative agent found. Two such studies have shown an increased risk in breast cancer in office workers exposed to strong magnetic fields. However, for many cancer clusters, although a common aetiology was not proven, it could not be discounted, highlighting the difficulty in definitively associating a causal agent to a cancer cluster. Nevertheless, in 2004 when staff at the Australian Broadcasting Corporation (ABC) premises in Toowong, Queensland, raised concerns about the number of breast cancer cases occurring among female employees at that site, investigations were initiated. In 2005, two studies attempted to evaluate the likelihood of the ABC cancer cluster and to identify whether personal breast cancer risk factors or workplace exposure to electromagnetic fields were possible causal agents, but no firm conclusions could be drawn. When an additional patient was diagnosed with breast cancer in 2006, an independent panel was constituted for a more systematic inquiry. That review included 550 women employed fullor part-time during the period from 1 January 1994 to 30 June 2006. A total of 13 women were diagnosed with breast cancer, with a possible additional case, and 10 of these were diagnosed while still working at the Toowong studio. The expected number based on the Queensland rate of breast cancer was 1.6 cases. The likelihood of this occurring by chance was estimated at approximately one in a million and this represented a greater than 6-fold increased risk compared to the general female population. No specific cause of the cluster was identified, despite measurements of electromagnetic fields, ionising radiation, and chemical contamination of water and soil, as well as traditional breast cancer risk factors. In an attempt to elucidate the nature of this cluster, we instigated a review of the biology of the ABC patients’ breast tumours. Breast cancer is a heterogeneous disease composed of several molecular pathology subtypes which may arise via different biological pathways. The working hypothesis was that a comparison to molecular subtypes may show that the tumours within the cancer cluster would have more similar morphological and/or molecular characteristics than those seen among tumours from a comparison group, and that this might reflect a common exposure to an unknown factor which might, in the long term, help to identify a novel breast cancer risk factor. This study also included an analysis of viral agents as there are some data to suggest a role for a retrovirus like mouse mammary tumour virus (MMTV) in breast cancer. Fourteen patients from the ABC breast cancer cluster were contacted and consent was obtained to study their tumours.