Stephen G. Braye

Decreased expression of key tumour suppressor microRNAs is associated with lymph node metastases in triple negative breast cancer

BackgroundBreast cancer is the most common malignancy that develops in women, responsible for the highest cancer-associated death rates. Triple negative breast cancers represent an important subtype that have an aggressive clinical phenotype, are associated with a higher likelihood of metastasis and are not responsive to current targeted therapies. miRNAs have emerged as an attractive candidate for molecular biomarkers and treatment targets in breast cancer, but their role in the progression of triple negative breast cancer remains largely unexplored.MethodsThis study has investigated miRNA expression profiles in 31 primary triple negative breast cancer cases and in 13 matched lymph node metastases compared with 23 matched normal breast tissues to determine miRNAs associated with the initiation of this disease subtype and those associated with its metastasis.Results71 miRNAs were differentially expressed in triple negative breast cancer, the majority of which have previously been associated with breast cancer, including members of the miR-200 family and the miR-17-92 oncogenic cluster, suggesting that the majority of miRNAs involved in the initiation of triple negative breast cancer are not subtype specific. However, the repertoire of miRNAs expressed in lymph node negative and lymph node positive triple negative breast cancers were largely distinct from one another. In particular, miRNA profiles associated with lymph node negative disease tended to be up-regulated, while those associated with lymph node positive disease were down-regulated and largely overlapped with the profiles of their matched lymph node metastases. From this, 27 miRNAs were identified that are associated with metastatic capability in the triple negative breast cancer subtype.ConclusionsThese results provide novel insight into the repertoire of miRNAs that contribute to the initiation of and progression to lymph node metastasis in triple negative breast cancer and have important implications for the treatment of this breast cancer subtype.

The expression of Dicer and Drosha in matched normal tissues, tumours and lymph node metastases in triple negative breast cancer

BackgroundBreast cancer is the most common malignancy in women world-wide. Triple negative breast cancer (TNBC) is a highly aggressive subtype that lacks expression of hormone receptors for estrogen, progesterone and human epidermal growth factor 2; and is associated with a high propensity for metastatic spread. Several studies have identified critical roles for microRNAs in breast cancer, but the role of two critical enzymes involved in microRNA biogenesis, Dicer and Drosha, is not well understood, particularly with respect to metastatic progression in this subtype.MethodsWe examined the expression of Dicer and Drosha in a series of invasive 35 TNBCs with matched normal adjacent tissues (n = 18) and lymph node metastases (n = 15) using semi-quantitative real time RT-PCR. The relationship of their expression with clinical features including age at diagnosis, lymph node positivity and tumour size was analysed.ResultsWe report that Dicer was significantly decreased while Drosha was significantly increased in tumours when compared to normal adjacent tissues. While there was no difference in Drosha expression in lymph node metastases when compared to the primary tumour, Dicer was significantly increased. There was no correlation between the expression of either Dicer or Drosha to age at diagnosis, lymph node positivity and tumour size.ConclusionsIn conclusion, Dicer and Drosha are dysregulated in TNBC and matched lymph node metastases however, the clinical relevance of this is still not known. The altered expression of Dicer and Drosha may serve as markers for disrupted miRNA biogenesis in TNBC.

Diagnostic ‘errors’ in anatomical pathology: relevance to Australian laboratories

&NA; Failure to recognise that anatomical pathology diagnosis is a process of cognitive interpretation of the morphological features present in a small tissue sample has led to the public misperception that the process is infallible. The absence of a universally accepted definition of diagnostic error makes comparison of error rates impossible and one large study of laboratories in the United States shows a significant error rate of about 5%, most of which have no major impact on patient management. A recent review of the work of one pathologist in New South Wales confirms a lack of appreciation in medical administration that variable diagnostic thresholds result in an inherent fallibility of anatomical pathology diagnoses. The outcome of the review emphasises the need to educate both public and non‐pathology colleagues of the nature of our work and brings into consideration the requirement to establish baseline error rates for Australian laboratories and the role of the Royal College of Pathologists of Australasia (RCPA) in developing fair and unbiased protocols for review of diagnostic errors. The responsibility of ensuring that diagnostic error rates are kept to the minimum is a shared one. Area health services must play their part by seeking to ensure that pathologists in any laboratory are not overworked and have adequate support and back‐up from pathologists with expertise in specialised areas. It has been clearly enunciated by the Royal College of Pathologists in the United Kingdom that it is not safe for any histopathology service to be operated single‐handedly by one histopathologist. Service managers and clinicians have to understand that country pathologists cannot provide the full range and depth of pathology expertise in the many clinical subspecialty areas that are often practised in non‐metropolitan areas. Attending clinicians share the responsibility of accepting proffered pathology diagnoses only if it conforms to the clinical context. Pathology laboratories must continue to develop and maintain best‐practice protocols and conduct periodic reviews of diagnosis, cytology‐histology concordance, frozen section/permanent section correlations, conference reviews, intra and interdepartmental consultations, participate in external quality assurance programs and maintain ongoing education for all laboratory staff.

Novel genes associated with lymph node metastasis in triple negative breast cancer

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and no targeted treatments. TNBC patients are more likely to develop metastases and relapse than patients with other breast cancer subtypes. We aimed to identify TNBC-specific genes and genes associated with lymph node metastasis, one of the first signs of metastatic spread. A total of 33 TNBCs were used; 17 of which had matched normal adjacent tissues available, and 15 with matched lymph node metastases. Gene expression microarray analysis was used to reveal genes that were differentially expressed between these groups. We identified and validated 66 genes that are significantly altered when comparing tumours to normal adjacent samples. Further, we identified 83 genes that are associated with lymph node metastasis and correlated these with miRNA-expression. Pathway analysis revealed their involvement in DNA repair, recombination and cell death, chromosomal instability and other known cancer-related pathways. Finally, four genes were identified that were specific for TNBC, of which one was associated with overall survival. This study has identified novel genes involved in LN metastases in TNBC and genes that are TNBC specific that may be used as treatment targets or prognostic indicators in the future.

The True Nature of Atypical Breast Cytology

Background: Atypical breast cytology is a poorly understood heterogeneous category with limited clinical utility but significant implications for patient management. Objective: To provide an insight into the true nature of atypical breast cytology in screening-detected (asymptomatic) and symptomatic settings, and find strategies for reducing the use of this diagnostic category. Materials and Methods: A total of 6,415 breast cytology samples were processed between January 2004 and December 2008. An atypical cytological diagnosis was rendered in 256 (4%) of the cases. A blind microscopic review of the atypical cases was conducted and results were correlated with subsequent histological and/or clinical outcomes. Results: Follow-up information by histology was available in 85.5%, by repeat fine-needle aspiration (FNA) in 3.5% and by imaging or clinical follow-up in 10.2% of the cases. Two patients (0.8%) were lost to follow-up. Of the 254 cases with follow-up, 62.6% were benign and 37.4% were malignant. The benign to malignant ratios were 1:1 and 2:1 in the screening and symptomatic groups, respectively. The atypical category in the screening population mostly yielded fat necrosis, complex sclerosing lesions and low- to intermediate-grade carcinoma on follow-up. The main outcomes in the symptomatic group were papilloma, fibroadenoma, ductal carcinoma in situ and lobular carcinoma. Preanalytical (suboptimal samples) factors were encountered in 34.8% and interpretative factors in 65.2% of the cases. Uncertainty about cellular morphology was attributed to such a diagnosis in 38 (14.8%) of the cases, architectural complexity in 137 (53.5%) and morphology and architecture in 70 (27.3%); 4.3% of cases were considered nondiagnostic. Conclusion: The atypical category is a necessary diagnosis but of limited use from a patient management perspective. Some preanalytical factors such as poor sample quality can be minimized by the involvement of cytopathologists in the FNA procedure. The use of the atypical category is partly dependent on the experience and confidence of the reporting pathologist. Assigning a case to this category is also likely to be unduly influenced by clinical or radiological findings. Our study indicates that the use of the atypical category can be reduced by up to 40% by appreciating these contributing factors. The practical utilization of the atypical category in breast cytology remains subjective and further study is required to identify useful objective criteria.

DNA methylation profile of triple negative breast cancer-specific genes comparing lymph node positive patients to lymph node negative patients

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with no targeted treatment available. Our previous study identified 38 TNBC-specific genes with altered expression comparing tumour to normal samples. This study aimed to establish whether DNA methylation contributed to these expression changes in the same cohort as well as disease progression from primary breast tumour to lymph node metastasis associated with changes in the epigenome. We obtained DNA from 23 primary TNBC samples, 12 matched lymph node metastases, and 11 matched normal adjacent tissues and assayed for differential methylation profiles using Illumina HumanMethylation450 BeadChips. The results were validated in an independent cohort of 70 primary TNBC samples. The expression of 16/38 TNBC-specific genes was associated with alteration in DNA methylation. Novel methylation changes between primary tumours and lymph node metastases, as well as those associated with survival were identified. Altered methylation of 18 genes associated with lymph node metastasis were identified and validated. This study reveals the important role DNA methylation plays in altered gene expression of TNBC-specific genes and lymph node metastases. The novel insights into progression of TNBC to secondary disease may provide potential prognostic indicators for this hard-to-treat breast cancer subtype.

Repair of UVB-induced DNA damage is reduced in melanoma due to low XPC and global genome repair

UVB exposure leads to DNA damage, which when unrepaired induces C>T transitions. These mutations are found throughout the melanoma genome, particularly in non-transcribed regions. The global genome repair (GGR) branch of nucleotide excision repair (NER) is responsible for repairing UV-induced DNA damage across non-transcribed and silent regions of the genome. This study aimed to examine the relationship between UVB and GGR in melanoma. DNA repair capacity and relative expression of NER in melanocytes and melanoma cell lines before and after treatment with UVB was quantified. Transcript expression from 196 melanomas was compared to clinical parameters including solar elastosis and whole transcriptome data collected. Melanoma cell lines showed significantly reduced DNA repair when compared to melanocytes, most significantly in the S phase of the cell cycle. Expression of GGR components XPC, DDB1 and DDB2 was significantly lower in melanoma after UVB. In the melanoma tumours, XPC expression correlated with age of diagnosis and low XPC conferred significantly poorer survival. The same trend was seen in the TCGA melanoma dataset. Reduced GGR in melanoma may contribute to the UV mutation spectrum of the melanoma genome and adds further to the growing evidence of the link between UV, NER and melanoma.

The microscopic complexities of C3 in breast cytology.

Background: Fine-needle aspiration (FNA) of difficult breast lesions often results in an atypical (C3) report. The assortment of outcomes generated by C3 reports varies widely, and this has given rise to different clinical management pathways. Objective: To identify and objectively assess microscopic features associated with atypical/C3 breast FNA cases. Materials and Methods: A total of 230 atypical breast FNAs were subjected to a blind microscopic rescreen using a range of robust qualitative and quantitative cytological criteria including cellularity, architectural qualities, cytomorphology and background features. A logistic regression with a receiver operating characteristic (ROC) curve and the resultant forward stepwise analysis were conducted to assess the results. This statistical testing was measured against malignant, benign proliferative and benign non-proliferative outcomes. Results: The malignant and benign proliferative outcomes showed a mixture of opposing protective and predictive individual cytological criteria. The stepwise analysis produced models demonstrating the best combination of individual cytological criteria for malignancy, proliferative and benign non-proliferative entities. In the malignancy model, discohesion, nuclear crowding within sheets, diminished numbers of bare bipolar nuclei and myoepithelial cells, the presence of tubules or necrosis and the absence of a cystic background were important features. The benign proliferative model suggested the same criteria but with the opposite implication and with the addition of several others, such as the presence of apocrine metaplasia, retained polarity and a speckled or coarse chromatin pattern. Age was a significant factor in malignant and proliferative outcomes. The benign non-proliferative stepwise analysis produced a model with fewer criteria (complex sheets, bare bipolar nuclei and a cystic background) limiting clinical application. Conclusion: Atypical/C3 breast cytology remains a legitimate reporting category. However, it is associated with a number of different histological outcomes. The incidence of the C3 category can be significantly reduced by controlling extrinsic factors and understanding the associated microscopic features.

Regulators of global genome repair do not respond to DNA damaging therapy but correlate with survival in melanoma.

Nucleotide excision repair (NER) orchestrates the repair of helix distorting DNA damage, induced by both ultraviolet radiation (UVR) and cisplatin. There is evidence that the global genome repair (GGR) arm of NER is dysfunctional in melanoma and it is known to have limited induction in melanoma cell lines after cisplatin treatment. The aims of this study were to examine mRNA transcript levels of regulators of GGR and to investigate the downstream effect on global transcript expression in melanoma cell lines after cisplatin treatment and in melanoma tumours. The GGR regulators, BRCA1 and PCNA, were induced in melanocytes after cisplatin, but not in melanoma cell lines. Transcripts associated with BRCA1, BRCA2, ATM and CHEK2 showed altered expression in melanoma cell lines after cisplatin treatment. In melanoma tumour tissue BRCA1 transcript expression correlated with poor survival and XPB expression correlated with solar elastosis levels. Taken together, these findings provide evidence of the mechanisms underlying NER deficiency in melanoma.

Can a familial gastrointestinal tumour syndrome be allelic with Waardenburg syndrome

Vilain RE, Dudding T, Braye SG, Groombridge C, Meldrum C, Spigelman AD, Ackland S, Ashman L, Scott RJ. Can a familial gastrointestinal tumour syndrome be allelic with Waardenburg syndrome?