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1.  FKBPL: a marker of good prognosis in breast cancer 
Oncotarget  2015;6(14):12209-12223.
FK506-binding protein-like (FKBPL) has established roles as an anti-tumor protein, with a therapeutic peptide based on this protein, ALM201, shortly entering phase I/II clinical trials. Here, we evaluated FKBPL's prognostic ability in primary breast cancer tissue, represented on tissue microarrays (TMA) from 3277 women recruited into five independent retrospective studies, using immunohistochemistry (IHC). In a meta-analysis, FKBPL levels were a significant predictor of BCSS; low FKBPL levels indicated poorer breast cancer specific survival (BCSS) (hazard ratio (HR) = 1.30, 95% confidence interval (CI) 1.14–1.49, p < 0.001). The prognostic impact of FKBPL remained significant after adjusting for other known prognostic factors (HR = 1.25, 95% CI 1.07–1.45, p = 0.004). For the sub-groups of 2365 estrogen receptor (ER) positive patients and 1649 tamoxifen treated patients, FKBPL was significantly associated with BCSS (HR = 1.34, 95% CI 1.13–1.58, p < 0.001, and HR = 1.25, 95% CI 1.04–1.49, p = 0.02, respectively). A univariate analysis revealed that FKBPL was also a significant predictor of relapse free interval (RFI) within the ER positive patient group, but it was only borderline significant within the smaller tamoxifen treated patient group (HR = 1.32 95% CI 1.05–1.65, p = 0.02 and HR = 1.23 95% CI 0.99–1.54, p = 0.06, respectively). The data suggests a role for FKBPL as a prognostic factor for BCSS, with the potential to be routinely evaluated within the clinic.
PMCID: PMC4494933  PMID: 25906750
FKBPL; breast cancer; biomarker; personalized medicine
2.  Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents 
Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.
PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n = 980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.
We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P = 1.29 × 10−4 and P = 0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis.
Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-015-0564-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4445529  PMID: 25902869
3.  The reliability of assessment of oestrogen receptor expression on needle core biopsy specimens of invasive carcinomas of the breast 
Journal of Clinical Pathology  2006;60(3):299-302.
To assess the reliability of assessment of oestrogen receptor expression on needle core biopsy specimens of invasive carcinomas of the breast. Previous studies have mostly been small, with a range of agreement from 62% to 100%.
Retrospective audit of 338 tumours surgically excised within 60 days of core biopsy, that had had oestrogen receptor assessed on both the core biopsy and tumour specimens. Surgical specimens were incised when fresh to ensure good fixation. External controls including a weakly positive tumour were included in each immunohistochemistry run.
Oestrogen receptor expression was bimodal, with H score in both specimens of either 0 or >50 in 96% of tumours. Using H score cut‐off of 10 for positivity, there was an agreement between core and excision in 334 of 338 tumours (98.8%). All discrepancies were between weakly positive and negative tumours. Intratumoral heterogeneity could explain the one tumour that was negative on core and positive on excision. H score tended to be slightly higher on core than excision (means 146 and 136). Better fixation on the core is the most likely explanation for this and for the three tumours that were positive on core and negative on excision. Repeat staining on tumours with discrepant results gave similar results in all except one case. An internal control was present in 97% of excisions and 55% of cores of oestrogen receptor‐negative tumours; the internal control stained positively in all except two sections.
Oestrogen receptor can be assessed reliably on needle core biopsies of invasive carcinomas of the breast.
PMCID: PMC1860557  PMID: 16731591
4.  DNA damage response markers are differentially expressed in BRCA-mutated breast cancers 
Cells have stringent DNA repair pathways that are specific for each different set of DNA lesions which is accomplished through the integration of complex array of proteins. However, BRCA-mutated breast cancer (BC) has defective DNA repair mechanisms. This study aims to investigate differential expression of a large panel of DNA repair markers to characterise DNA repair mechanisms in BRCA-associated tumours compared to sporadic tumours in an attempt to characterise these tumours in routine practice. Immunohistochemistry and tissue microarray technology were applied to a cohort of clinically annotated series of sporadic (n = 1849), BRCA1-mutated (n = 48), and BRCA2-mutated (n = 27) BC. The following DNA damage response (DDR) markers are used; BRCA1, BRCA2, RAD51, Ku70/Ku80, BARD, PARP1 (cleaved), PARP1 (non-cleaved), and P53 in addition to basal cytokeratins, ER, PR, and HER2. A significant proportion of BRCA1 tumours were positive for PARP1 (non-cleaved), and negative for BARD1 and RAD51 compared with sporadic BC. BRCA2 tumours were significantly positive for PARP1 (non-cleaved) compared with sporadic tumours. RAD51 was significantly higher in BRCA1 compared with BRCA2 tumours (p = 0.005). When BRCA1/2 BCs were compared to triple-negative (TN) sporadic tumours of the studied DDR proteins, BARD1 (p < 0.001), PARP1 (non-cleaved) (p < 0.001), and P53 (p = 0.002) remained significantly different in BRCA1/2 tumours compared with TN BC. DNA repair markers showed differential expression in BRCA-mutated tumours, with a substantial degree of disruption of DNA repair pathways in sporadic BC especially TN BC. DNA double-strand break (DSB) repair is assisted by PARP1 expression in BRCA-mutated tumours, whereas the loss of DSB repair via RAD51 is predominant in BRCA1 rather than BRCA2 BC.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-015-3306-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4344553  PMID: 25690937
DNA repair; DNA damage response; BRCA-mutated breast cancers
5.  A tumor DNA complex aberration index is an independent predictor of survival in breast and ovarian cancer 
Molecular Oncology  2015;9(1):115-127.
Complex focal chromosomal rearrangements in cancer genomes, also called “firestorms”, can be scored from DNA copy number data. The complex arm-wise aberration index (CAAI) is a score that captures DNA copy number alterations that appear as focal complex events in tumors, and has potential prognostic value in breast cancer. This study aimed to validate this DNA-based prognostic index in breast cancer and test for the first time its potential prognostic value in ovarian cancer. Copy number alteration (CNA) data from 1950 breast carcinomas (METABRIC cohort) and 508 high-grade serous ovarian carcinomas (TCGA dataset) were analyzed. Cases were classified as CAAI positive if at least one complex focal event was scored. Complex alterations were frequently localized on chromosome 8p (n = 159), 17q (n = 176) and 11q (n = 251). CAAI events on 11q were most frequent in estrogen receptor positive (ER+) cases and on 17q in estrogen receptor negative (ER−) cases. We found only a modest correlation between CAAI and the overall rate of genomic instability (GII) and number of breakpoints (r = 0.27 and r = 0.42, p < 0.001). Breast cancer specific survival (BCSS), overall survival (OS) and ovarian cancer progression free survival (PFS) were used as clinical end points in Cox proportional hazard model survival analyses. CAAI positive breast cancers (43%) had higher mortality: hazard ratio (HR) of 1.94 (95%CI, 1.62–2.32) for BCSS, and of 1.49 (95%CI, 1.30–1.71) for OS. Representations of the 70-gene and the 21-gene predictors were compared with CAAI in multivariable models and CAAI was independently significant with a Cox adjusted HR of 1.56 (95%CI, 1.23–1.99) for ER+ and 1.55 (95%CI, 1.11–2.18) for ER− disease. None of the expression-based predictors were prognostic in the ER− subset. We found that a model including CAAI and the two expression-based prognostic signatures outperformed a model including the 21-gene and 70-gene signatures but excluding CAAI. Inclusion of CAAI in the clinical prognostication tool PREDICT significantly improved its performance. CAAI positive ovarian cancers (52%) also had worse prognosis: HRs of 1.3 (95%CI, 1.1–1.7) for PFS and 1.3 (95%CI, 1.1–1.6) for OS. This study validates CAAI as an independent predictor of survival in both ER+ and ER− breast cancer and reveals a significant prognostic value for CAAI in high-grade serous ovarian cancer.
•The complex arm-wise aberration index (CAAI) captures focal complex DNA alterations.•Compared with other indices of genomic instability, CAAI adds unique information.•CAAI is validated as an independent prognostic marker in breast cancer (n = 1950).•Prognostic value of CAAI is independent of the 70- and 21-gene classifiers.•CAAI is a new independent prognostic marker in ovarian cancer.
PMCID: PMC4286124  PMID: 25169931
Breast cancer; Ovarian cancer; Prognostic markers; Biomarker; Genomics; Genomic instability; DNA copy number; BCSS, Breast cancer specific survival; CAAI, Complex arm-wise aberration index; CNA, Copy number alterations; ER, Estrogen receptor; HR, Hazard ratio; HGSOC, High-grade serous ovarian cancer; MIP, Molecular inversion probe; OS, Overall survival; PFS, Progression free survival
6.  Low calpain-9 is associated with adverse disease-specific survival following endocrine therapy in breast cancer 
BMC Cancer  2014;14:995.
The calpains are intracellular cysteine proteases that function in a variety of important cellular functions, including signalling, motility, apoptosis and survival. In breast cancer high calpain-1 and calpain-2 expression has been associated with adverse clinical outcome. Calpain-9 was thought to be exclusively expressed in the digestive tract; however recent studies have shown that this protein is also expressed in breast tissue.
We investigated the expression of calpain-9 in a large cohort of early stage breast cancer patients (n = 783) using immunohistochemistry on a tissue microarray. Patients had long-term follow-up information available for analysis.
Low expression of calpain-9 was associated with patients over 40 years of age (P = 0.025), smaller tumour size (P = 0.001), lower tumour stage (P = 0.009), a more favourable Nottingham Prognostic Index value (P = 0.002) and positive oestrogen receptor status (P = 0.014). Calpain-9 expression was not associated with survival in the total patient cohort, however low calpain-9 expression was associated with adverse survival in patients who received endocrine therapy (P = 0.033), which remained significant in multivariate Cox regression analysis accounting for potential confounding factors (hazard ratio (HR) = 0.56, 95% confidence interval (95% CI) = 0.36-0.89, P = 0.013). Low calpain-9 expression was also associated with adverse survival in patients with an intermediate Nottingham Prognostic Index value (P = 0.009), and remained so in multivariate analysis (HR = 0.54, 95% CI = 0.36-0.82, P = 0.003).
This study suggests that calpain-9 may play a role in breast cancer and that low expression is associated with poorer patient clinical outcome following endocrine therapy. Validation studies are warranted as determining expression of calpain-9 may provide important prognostic information.
PMCID: PMC4364321  PMID: 25539577
Calpain; Calpain-9; Breast cancer; Endocrine therapy
7.  Cytoplasmic expression of p27kip1 is associated with a favourable prognosis in colorectal cancer patients 
AIM: To evaluate the prognostic significance of p27kip1 in colorectal cancer patients.
METHODS: Cytoplasmic and nuclear p27kip1 expression was evaluated in 418 colorectal cancers using tissue microarrays. Data were associated with known patient and tumor variables and long-term patient outcomes, providing further insight into the mechanisms by which p27kip1 may influence tumor development.
RESULTS: Nuclear and cytoplasmic p27Kip1 expressions were detected in 59% and 19% of tumors respectively. Cytoplasmic p27Kip1 was almost invariably associated with positive nuclear p27Kip1 expression. Neither case correlated with known clinical or pathological variables, including tumor stage, grade or extramural vascular invasion. Furthermore, nuclear p27kip1 expression had no impact on survival. However, we identified a significant correlation between expression of cytoplasmic p27kip1 and longer disease-specific survival times. On multivariate analysis, TNM stage and extramural vascular invasion were highly significant independent prognostic factors, with positive cytoplasmic p27 expression showing a trend towards improved patient survival (P = 0.059).
CONCLUSION: These findings support the recent evidence that cytoplasmic p27kip1 has a distinct and important biological role that can influence tumor outcome.
PMCID: PMC4088137  PMID: 17072952
Colorectal cancer; Tissue microarray; p27Kip1; Prognostic factor
8.  Biological and clinical significance of PARP1 protein expression in breast cancer 
Poly(ADP-ribose) polymerase-1 (PARP1) is a key facilitator of DNA repair. PARP inhibitors have gained recent attention as promising therapeutic agents for the treatment of solid tumours including breast cancer (BC). However, the biological and clinical significance of PARP1 expression in BC and its role in DNA-damage response (DDR) remain to be defined. We investigated the expression of PARP1 expression, cleaved (PARP1c) and non-cleaved (PAR1nc) forms, in a large and well-characterised cohort of clinically annotated stage I–III operable BCs (n = 1,269) and 43 BRCA1-mutated BCs using immunohistochemistry. PARP1 expression was correlated to clinicopathological variables, outcome and expression of other key DNA repair proteins (BRCA1, RAD51, Ku70/80, PIASγ and CHK1). Expression of PARP1 was exclusively nuclear. 49 and 85 % of sporadic BC showed expression PARP1nc and PARP1c, respectively. In BRCA1-mutated tumours, PARP1nc/PARP1c was highly expressed (95 and 79 %, respectively). PARP1nc expression was positively associated with premenopausal younger age patients, larger size and higher tumour grade. PARP1 was positively associated with DDR-proteins; RAD51, BRCA1, CHK1 and PIASγ (p < 0.001). Negative association was found between PARP1nc and Ki67. PARP1c was associated with ER (p < 0.001). Different associations between PARP1 and DDR-proteins were observed when stratified based on ER/BRCA1 status. PARP1 was not an independent predictor of outcome in sporadic or BRCA1-mutated BC. Our results demonstrate a potential biological role for PARP1c and PARP1nc in DNA repair in BC based on the significant association with other key DNA damage repair proteins. These associations were not restricted to ER-negative or triple-negative subgroup.
PMCID: PMC4308637  PMID: 25528020
PARP1; Breast cancer; DNA repair gene defects; BRCA1; Biology; Clinical significance
9.  Involvement of metformin and AMPK in the radioresponse and prognosis of luminal versus basal-like breast cancer treated with radiotherapy 
Oncotarget  2014;5(24):12936-12949.
Metformin is under evaluation as a potential anticancer agent. Expression of total and phospho(Thr172)-adenosine monophosphate-activated kinase-α (AMPKα and pAMPKα(Thr172) respectively), a main metformin target, was examined in radiotherapy treated breast cancers and metformin's ability to modulate Trx system expression and breast cancer radiosensitivity evaluated in vitro.
AMPKα and pAMPKα(Thr172) expression was assessed using a discovery (n=166) and validation cohort (n=609). Metformin's role in regulating radioresponse, and Trx family expression, was examined via clonogenic assays and Western blots. Intracellular reactive oxygen species (ROS) levels, cell cycle progression and apoptosis were assessed by flow cytometry.
High AMPKα expression associated with improved local recurrence-free (P=0.019), relapse-free (P=0.016) and breast cancer-specific survival (P=0.000065) and was, from multivariate analysis, an independent prognostic factor from the discovery cohort. From the validation cases AMPKα expression associated with relapse-free and breast cancer-specific survival in luminal breast cancers. Metformin substantially increased radiosensitivity, intracellular ROS levels and reduced Trx expression, in luminal breast cancer cells, but had little effect on basal phenotype cells.
In conclusion, high AMPKα expression associates with improved prognosis, especially in luminal breast cancer. Metformin preferentially radiosensitises luminal breast cancer cells, potentially due to alterations to intracellular ROS levels via modulation of Trx family protein expression.
PMCID: PMC4350336  PMID: 25427448
metformin; AMPK; radiosensitivity; breast cancer; luminal phenotype
10.  Clinicopathological Significance of ATM-Chk2 Expression in Sporadic Breast Cancers: a Comprehensive Analysis in Large Cohorts1 
Neoplasia (New York, N.Y.)  2014;16(11):982-991.
ATM-Chk2 network is critical for genomic stability, and its deregulation may influence breast cancer pathogenesis. We investigated ATM and Chk2 protein levels in two cohorts [cohort 1 (n = 1650) and cohort 2 (n = 252)]. ATM and Chk2 mRNA expression was evaluated in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1950). Low nuclear ATM protein level was significantly associated with aggressive breast cancer including larger tumors, higher tumor grade, higher mitotic index, pleomorphism, tumor type, lymphovascular invasion, estrogen receptor (ER)−, PR −, AR −, triple-negative, and basal-like phenotypes (Ps < .05). Breast cancer 1, early onset negative, low XRCC1, low SMUG1, high FEN1, high MIB1, p53 mutants, low MDM2, low Bcl-2, low p21, low Bax, high CDK1, and low Chk2 were also more frequent in tumors with low nuclear ATM level (Ps < .05). Low ATM protein level was significantly associated with poor survival including in patients with ER-negative tumors who received adjuvant anthracycline or cyclophosphamide, methotrexate, and 5-fluorouracil–based adjuvant chemotherapy (Ps < .05). Low nuclear Chk2 protein was likely in ER −/PR −/AR −; HER-2 positive; breast cancer 1, early onset negative; low XRCC1; low SMUG1; low APE1; low polβ; low DNA-PKcs; low ATM; low Bcl-2; and low TOPO2A tumors (P < .05). In patients with ER + tumors who received endocrine therapy or ER-negative tumors who received chemotherapy, nuclear Chk2 levels did not significantly influence survival. In p53 mutant tumors, low ATM (P < .000001) or high Chk2 (P < .01) was associated with poor survival. When investigated together, low-ATM/high-Chk2 tumors have the worst survival (P = .0033). Our data suggest that ATM-Chk2 levels in sporadic breast cancer may have prognostic and predictive significance.
PMCID: PMC4240925  PMID: 25425972
11.  Biological Characteristics and Clinical Outcome of Triple Negative Primary Breast Cancer in Older Women – Comparison with Their Younger Counterparts 
PLoS ONE  2014;9(7):e100573.
Triple negative (ER, PgR and HER2 negative) breast cancers (TNBCs) are often considered as a poor prognostic phenotype. There is dearth of evidence showing the prevalence and biological behaviour of TNBCs in older women. This study aimed to analyse their biological characteristics in comparison with a well characterised younger series from a single centre with long term clinical follow-up. Over 37 years (1973–2010), 1,758 older (≥70 years) women with early operable (<5 cm) primary breast cancer were managed in a dedicated clinic and have complete clinical information available. Of these 813 patients underwent primary surgery and 575 had good quality tumour samples available for tissue microarray analysis using indirect immunohistochemistry. A total of 127 patients (22.1%) had TNBCs and full biological analysis of 15 biomarkers was performed. The results were compared with those of their younger (<70 years) counterparts 342 (18.9%) from a previously characterised, consecutive series of primary breast cancer treated in the same unit (1986–1998). The 127 older patients with TNBCs showed lower rates of Ki67 and CK 7/8 positivity and high rates of bcl2 and CK18 positivity when compared with their younger counterparts (p<0.05). There was no significant difference in the long term clinical outcome between the two age groups, despite the fact that 47% of the younger patients had adjuvant chemotherapy, while none in the older cohort received such treatment. EGFR, axillary stage and pathological size showed prognostic significance in older women with TNBCs on univariate analysis. Despite not having received adjuvant chemotherapy, the older series had clinical outcome similar to the younger patients almost half of whom had chemotherapy. This appears to be related to other biomarkers (in addition to ER/PgR/HER2) eg Ki67, bcl2 and cytokeratins which have different expression patterns influencing prognosis.
PMCID: PMC4085072  PMID: 24999743
12.  Therapeutic Targeting of Integrin αvβ6 in Breast Cancer 
Integrin αvβ6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer.
Protein expression of integrin subunit beta6 (β6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student’s t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni’s Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided.
High expression of either the mRNA or protein for the integrin subunit β6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of β6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts.
Targeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients.
PMCID: PMC4151855  PMID: 24974129
13.  Immunoglobulin free light chains are biomarkers of poor prognosis in basal-like breast cancer and are potential targets in tumor-associated inflammation 
Oncotarget  2014;5(10):3159-3167.
Inflammation is an important component of various cancers and its inflammatory cells and mediators have been shown to have prognostic potential. Tumor-infiltrating mast cells can promote tumor growth and angiogenesis, but the mechanism of mast cell activation is unclear. In earlier studies, we demonstrated that immunoglobulin free light chains (FLC) can trigger mast cells in an antigen-specific manner. Increased expression of FLC was observed within stroma of various human cancers including those of breast, colon, lung, pancreas, kidney and skin, and FLC expression co-localized with areas of mast cell infiltration. In a large cohort of breast cancer patients, FLC expression was shown associated with basal-like cancers with an aggressive phenotype. Moreover, lambda FLC was found expressed in areas of inflammatory infiltration and its expression was significantly associated with poor clinical outcome. Functional importance of FLCs was shown in a murine B16F10 melanoma model, where inhibition of FLC-mediated mast cell activation strongly reduced tumor growth. Collectively, this study identifies FLCs as a ligand in the pro-tumorigenic activation of mast cells. Blocking this pathway may open new avenues for the inhibition of tumor growth, while immunohistochemical staining of FLC may be helpful in the diagnosis and prognosis of cancer.
PMCID: PMC4102799  PMID: 24931643
immunoglobulin free light chain; mast cell; biomarker; inflammation; tumor progression
14.  Histological grading of breast cancer on needle core biopsy: the role of immunohistochemical assessment of proliferation 
Histopathology  2010;57(2):212-219.
Histological grade assessed on needle core biopsy (NCB) moderately concurs with the grade in the surgical excision specimen (SES) (κ-values between 0.35 and 0.65). A major cause of the discrepancy is underestimation of mitoses in the NCB specimen. The aim was to determine the best method of assessing proliferation on NCB.
Methods and results
Proliferative activity of 101 invasive carcinomas of the breast on NCB and SES was assessed using mitotic counts on routine haematoxylin and eosin (H&E) sections and immunohistochemical markers Mib-1 and phosphorylated histone H3 (PPH3). H&E mitotic count in SES was considered as the gold standard. H&E mitotic count was found to be underestimated on NCB when compared with that in SES (P < 0.001), but no significant difference was detected between NCB and SES regarding Mib-1 (P = 0.13) or PPH3 (P = 0.073). Using receiver–operating characteristic curve, Mib-1 on NCB was found to agree with the gold standard significantly better than routine H&E on NCB.
Immunohistochemical markers in NCB showed better concordance with H&E mitotic count in SES (gold standard) than routine H&E mitotic count in NCB. Further refinement of cut-offs and scoring methods is needed.
PMCID: PMC3564399  PMID: 20716163
breast neoplasm; histological grading; needle core biopsy
15.  HER2/HER3 heterodimers and p21 expression are capable of predicting adjuvant trastuzumab response in HER2+ breast cancer 
Human epidermal growth factor receptor 2 (HER2) plays an important role in breast cancer progression and provides predictive information for response to targeted therapy including trastuzumab although this is limited. Downstream pathways, such as PI3K/Akt, are associated with HER2/HER3 heterodimerization promoting survival and proliferation amongst cancer cells. Thus, patient outcome and trastuzumab therapy effectiveness might be further characterised by HER2/HER3 dimerisation and its signalling pathways. HER2/HER3 dimerisation status was assessed, using chromogenic in situ Proximity Ligation Assay, in two breast cancer series: early stage primary breast cancer, including 224 HER2+ patients that were not submitted to trastuzumab, and HER2+ breast cancer where patients were treated with adjuvant trastuzumab (n = 143). Levels of biomarkers including PI3K, pAKT, ER, PgR, HER3, BCL2, p53, PTEN and p21 were measured using immunohistochemistry. Levels of HER2/HER3 heterodimers were compared with biomarker expression and patient outcome. An association between high levels of HER2/HER3 dimerisation and absence of hormone receptors, ER and PgR, was observed. We further show for the first time the presence of HER2/HER3 heterodimers and the loss of p21 expression in HER2+ breast cancer predicts a significantly poorer outcome when submitted to adjuvant trastuzumab. Breast cancer patients that reveal high levels of HER2/HER3 dimerisation and loss of p21 are associated with poor survival prognosis in patients with HER2+ breast cancer treated with adjuvant trastuzumab. Further quantification analysis of HER dimer/ligand complexes and downstream signalling pathways will begin to unravel the complex associations with patient outcome and its relationship with sensitivity to targeted treatment.
PMCID: PMC3984415  PMID: 24706169
Breast cancer; Trastuzumab; p21; HER2/HER3 heterodimers
16.  A methodology to ensure and improve accuracy of Ki67 labelling index estimation by automated digital image analysis in breast cancer tissue 
Immunohistochemical Ki67 labelling index (Ki67 LI) reflects proliferative activity and is a potential prognostic/predictive marker of breast cancer. However, its clinical utility is hindered by the lack of standardized measurement methodologies. Besides tissue heterogeneity aspects, the key element of methodology remains accurate estimation of Ki67-stained/counterstained tumour cell profiles. We aimed to develop a methodology to ensure and improve accuracy of the digital image analysis (DIA) approach.
Tissue microarrays (one 1-mm spot per patient, n = 164) from invasive ductal breast carcinoma were stained for Ki67 and scanned. Criterion standard (Ki67-Count) was obtained by counting positive and negative tumour cell profiles using a stereology grid overlaid on a spot image. DIA was performed with Aperio Genie/Nuclear algorithms. A bias was estimated by ANOVA, correlation and regression analyses. Calibration steps of the DIA by adjusting the algorithm settings were performed: first, by subjective DIA quality assessment (DIA-1), and second, to compensate the bias established (DIA-2). Visual estimate (Ki67-VE) on the same images was performed by five pathologists independently.
ANOVA revealed significant underestimation bias (P < 0.05) for DIA-0, DIA-1 and two pathologists’ VE, while DIA-2, VE-median and three other VEs were within the same range. Regression analyses revealed best accuracy for the DIA-2 (R-square = 0.90) exceeding that of VE-median, individual VEs and other DIA settings. Bidirectional bias for the DIA-2 with overestimation at low, and underestimation at high ends of the scale was detected. Measurement error correction by inverse regression was applied to improve DIA-2-based prediction of the Ki67-Count, in particular for the clinically relevant interval of Ki67-Count < 40%. Potential clinical impact of the prediction was tested by dichotomising the cases at the cut-off values of 10, 15, and 20%. Misclassification rate of 5-7% was achieved, compared to that of 11-18% for the VE-median-based prediction.
Our experiments provide methodology to achieve accurate Ki67-LI estimation by DIA, based on proper validation, calibration, and measurement error correction procedures, guided by quantified bias from reference values obtained by stereology grid count. This basic validation step is an important prerequisite for high-throughput automated DIA applications to investigate tissue heterogeneity and clinical utility aspects of Ki67 and other immunohistochemistry (IHC) biomarkers.
PMCID: PMC4053156  PMID: 24708745
17.  DACH1: Its Role as a Classifier of Long Term Good Prognosis in Luminal Breast Cancer 
PLoS ONE  2014;9(1):e84428.
Oestrogen receptor (ER) positive (luminal) tumours account for the largest proportion of females with breast cancer. Theirs is a heterogeneous disease presenting clinical challenges in managing their treatment. Three main biological luminal groups have been identified but clinically these can be distilled into two prognostic groups in which Luminal A are accorded good prognosis and Luminal B correlate with poor prognosis. Further biomarkers are needed to attain classification consensus. Machine learning approaches like Artificial Neural Networks (ANNs) have been used for classification and identification of biomarkers in breast cancer using high throughput data. In this study, we have used an artificial neural network (ANN) approach to identify DACH1 as a candidate luminal marker and its role in predicting clinical outcome in breast cancer is assessed.
Materials and methods
A reiterative ANN approach incorporating a network inferencing algorithm was used to identify ER-associated biomarkers in a publically available cDNA microarray dataset. DACH1 was identified in having a strong influence on ER associated markers and a positive association with ER. Its clinical relevance in predicting breast cancer specific survival was investigated by statistically assessing protein expression levels after immunohistochemistry in a series of unselected breast cancers, formatted as a tissue microarray.
Strong nuclear DACH1 staining is more prevalent in tubular and lobular breast cancer. Its expression correlated with ER-alpha positive tumours expressing PgR, epithelial cytokeratins (CK)18/19 and ‘luminal-like’ markers of good prognosis including FOXA1 and RERG (p<0.05). DACH1 is increased in patients showing longer cancer specific survival and disease free interval and reduced metastasis formation (p<0.001). Nuclear DACH1 showed a negative association with markers of aggressive growth and poor prognosis.
Nuclear DACH1 expression appears to be a Luminal A biomarker predictive of good prognosis, but is not independent of clinical stage, tumour size, NPI status or systemic therapy.
PMCID: PMC3879319  PMID: 24392136
18.  Cell Proliferation (KI-67) Expression Is Associated with Poorer Prognosis in Nigerian Compared to British Breast Cancer Women 
ISRN Oncology  2013;2013:675051.
Background. Black women with breast cancer (BC) in Nigeria have higher mortality rate compared with British women. This study investigated prognostic features of cell proliferation biomarker (Ki-67) in Nigerian breast cancer women. Materials and Methods. The protein expression of Ki-67 was investigated in series of 308 Nigerian women, prepared as a tissue microarray (TMA), using immunohistochemistry. Clinic-pathological parameters, biomarkers, and patient outcome of tumours expressing Ki-67 in Nigerian women were correlated with UK grade-matched series. Results. A significantly larger proportion of breast tumours from Nigerian women showed high Ki-67 expression. Those tumours were significantly correlated with negative expression of the steroid hormone receptors (ER and PgR), p21, p27, E-cadherin, BRCA-1, and Bcl-2 (all P < 0.001), but positively associated with EGFR (P = 0.003), p53, basal cytokeratins: CK56, CK14, triple negative, and basal phenotype using Nielsen's classification (all P < 0.001) compared to UK women. Multivariate analyses showed that race was also associated with BCSS independent of tumour size, lymph node status, and ER status. Conclusion. Ki-67 expression was observed to have contributed to the difference in the BCSS in Nigerian compared with British BC women. Therefore, targeting Ki-67 in the indigenous black women with BC might improve the patient outcome in the black women with BC.
PMCID: PMC3649293  PMID: 23691362
19.  A randomized trial to assess the biological activity of short-term (pre-surgical) fulvestrant 500 mg plus anastrozole versus fulvestrant 500 mg alone or anastrozole alone on primary breast cancer 
Fulvestrant shows dose-dependent biological activity. Greater estrogen-receptor (ER) blockade may feasibly be achieved by combining fulvestrant with anastrozole. This pre-surgical study compared fulvestrant plus anastrozole versus either agent alone in patients with ER-positive breast cancer.
In this double-blind, multicenter trial, 121 patients received fulvestrant 500 mg on Day 1 plus anastrozole 1 mg/day for 14 to 21 days (F + A); fulvestrant plus anastrozole placebo (F); or fulvestrant placebo plus anastrozole (A), 2 to 3 weeks before surgery. ER, progesterone-receptor (PgR) and Ki67 expression were determined from tumor biopsies before treatment and at surgery.
A total of 103 paired samples were available (F, n = 35; F+A, n = 31; A, n = 37). All treatments significantly reduced mean ER expression from baseline (F: -41%, P = 0.0001; F + A: -39%, P = 0.0001; A: -13%, P = 0.0034). F and F + A led to greater reductions in ER versus A (both P = 0.0001); F + A did not lead to additional reductions versus F. PgR and Ki67 expression were significantly reduced with all treatments (means were -34% to -45%, and -75% to -85%, respectively; all P = 0.0001), with no differences between groups.
In this short-term study, all treatments reduced ER expression, although F and F + A showed greater reductions than A. No significant differences were detected between the treatment groups in terms of PgR and Ki67 expression. No additional reduction in tumor biomarkers with combination treatment was observed, suggesting that F + A is unlikely to have further clinical benefit over F alone.
Trial registration NCT00259090.
PMCID: PMC3672778  PMID: 23497452
20.  Cytoplasmic localization of alteration/deficiency in activation 3 (ADA3) predicts poor clinical outcome in breast cancer patients 
Transcriptional activation by estrogen receptor (ER) is a key step to breast oncogenesis. Given previous findings that ADA3 is a critical component of HAT complexes that regulate ER function and evidence that overexpression of other ER coactivators such as SRC-3 is associated with clinical outcomes in breast cancer, the current study was designed to assess the potential significance of ADA3 expression/localization in human breast cancer patients. In this study, we analyzed ADA3 expression in breast cancer tissue specimens and assessed the correlation of ADA3 staining with cancer progression and patient outcome. Tissue microarrays prepared from large series of breast cancer patients with long-term follow-ups were stained with anti-ADA3 monoclonal antibody using immunohistochemistry. Samples were analyzed for ADA3 expression followed by correlation with various clinicopathological parameters and patients’ outcomes. We report that breast cancer specimens show predominant nuclear, cytoplasmic, or mixed nuclear + cytoplasmic ADA3 staining patterns. Predominant nuclear ADA3 staining correlated with ER+ status. While predominant cytoplasmic ADA3 staining negatively correlated with ER+ status, but positively correlated with ErbB2, EGFR, and Ki67. Furthermore, a positive correlation of cytoplasmic ADA3 was observed with higher histological grade, mitotic counts, Nottingham Prognostic Index, and positive vascular invasion. Patients with nuclear ADA3 and ER positivity have better breast cancer specific survival and distant metastasis free survival. Significantly, cytoplasmic expression of ADA3 showed a strong positive association with reduced BCSS and DMFS in ErbB2+/EGFR+ patients. Although in multivariate analyses ADA3 expression was not an independent marker of survival, predominant nuclear ADA3 staining in breast cancer tissues correlates with ER+ expression and together serves as a marker of good prognosis, whereas predominant cytoplasmic ADA3 expression correlates with ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Thus, ADA3 cytoplasmic localization together with ErbB2+/EGFR+ status may serve as better prognostic marker than individual proteins to predict survival of patients.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-012-2363-3) contains supplementary material, which is available to authorized users.
PMCID: PMC3552361  PMID: 23288344
ADA3; STAGA; ATAC; Coactivator; ErbB2; ER; PR; Breast cancer
21.  Differential oestrogen receptor binding is associated with clinical outcome in breast cancer 
Nature  2012;481(7381):389-393.
Oestrogen receptor-α (ER) is the defining and driving transcription factor in the majority of breast cancers and its target genes dictate cell growth and endocrine response, yet genomic understanding of ER function has been restricted to model systems1-3. We now map genome-wide ER binding events, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), in primary breast cancers from patients with different clinical outcome and in distant ER positive (ER+) metastases. We find that drug resistant cancers still have ER-chromatin occupancy, but that ER binding is a dynamic process, with the acquisition of unique ER binding regions in tumours from patients that are likely to relapse. The acquired, poor outcome ER regulatory regions observed in primary tumours reveal gene signatures that predict clinical outcome in ER+ disease exclusively. We find that the differential ER binding programme observed in tumours from patients with poor outcome is not due to the selection of a rare subpopulation of cells, but is due to the FoxA1-mediated reprogramming of ER binding on a rapid time scale. The parallel redistribution of ER and FoxA1 cis-regulatory elements in drug resistant cellular contexts is supported by histological co-expression of ER and FoxA1 in metastatic samples. By establishing transcription factor mapping in primary tumour material, we show that there is plasticity in ER binding capacity, with distinct combinations of cis-regulatory elements linked with the different clinical outcomes.
PMCID: PMC3272464  PMID: 22217937
22.  Objective assessment of blood and lymphatic vessel invasion and association with macrophage infiltration in cutaneous melanoma 
Modern Pathology  2011;25(4):493-504.
The aims of this study were to investigate the role of vascular invasion (blood and lymphatic), vessel density and the presence of tumour-associated macrophages as prognostic markers in 202 cutaneous melanoma patients. Sections of primary melanoma were stained with lymphatic-specific antibody D2-40 to assess lymphatic vessel invasion and density in intratumoural and peritumoural areas; an antibody against endothelial marker CD34 was used to determine blood vessel invasion and density, and an antibody against CD68 was used to determine macrophage counts. Immunohistochemically determined vascular invasion (combined blood and lymphatic) was compared with that determined using haematoxylin and eosin (H&E) staining. The use of immunohistochemistry increased detection of vascular invasion from 8–30% of patients, and histological exam of H&E-stained tissue was associated with a false positive rate of 64%. Lymphatic vessel invasion occurred at a much higher frequency than blood vessel invasion (27 and 4% of patients, respectively). Although immunohistochemically detected vessel invasion was significantly associated with histological markers of adverse prognosis, such as increased Breslow thickness, ulceration and mitotic rate (all P<0.001), no associations with relapse-free or overall survival were observed. High macrophage counts were significantly associated with markers of aggressive disease, such as Breslow thickness, ulceration and mitotic rate (P<0.001, P<0.001, P=0.005, respectively), and lymphatic vessel invasion and high microvessel density (P=0.002 and P=0.003, respectively). These results suggest that vascular invasion is more accurately detected using immunohistochemistry and occurs predominantly via lymphatic vessels. The association of vessel characteristics with histological characteristics of the primary melanoma provides evidence for their biological importance in melanoma, but that they were not associated with clinical outcome attests to the value of existing histological prognostic biomarkers. We note that a high macrophage count may be associated with neovascularisation and primary tumour growth, and may also promote invasion through lymphatic vessels.
PMCID: PMC3318158  PMID: 22080065
D2-40; melanoma; tumour associated macrophages; vascular invasion; vessel density
23.  Overexpression of a novel cell cycle regulator ecdysoneless in breast cancer: a marker of poor prognosis in HER2/neu-overexpressing breast cancer patients 
Uncontrolled proliferation is one of the hallmarks of breast cancer. We have previously identified the human Ecd protein (human ortholog of Drosophila Ecdysoneless, hereafter called Ecd) as a novel promoter of mammalian cell cycle progression, a function related to its ability to remove the repressive effects of Rb-family tumor suppressors on E2F transcription factors. Given the frequent dysregulation of cell cycle regulatory components in human cancer, we used immunohistochemistry of paraffin-embedded tissues to examine Ecd expression in normal breast tissue versus tissues representing increasing breast cancer progression. Initial studies of a smaller cohort without outcomes information showed that Ecd expression was barely detectable in normal breast tissue and in hyperplasia of breast, but high levels of Ecd were detected in benign breast hyperplasia, ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDCs) of the breast. In this cohort of 104 IDC patients, Ecd expression levels showed a positive correlation with higher grade (P = 0.04). Further analyses of Ecd expression using a larger, independent cohort (954) confirmed these results, with a strong positive correlation of elevated Ecd expression with higher histological grade (P = 0.013), mitotic index (P = 0.032), and Nottingham Prognostic Index score (P = 0.014). Ecd expression was positively associated with HER2/neu (P = 0.002) overexpression, a known marker of poor prognosis in breast cancer. Significantly, increased Ecd expression showed a strong positive association with shorter breast cancer specific survival (BCSS) (P = 0.008) and disease-free survival (DFS) (P = 0.003) in HER2/neu overexpressing patients. Taken together, our results reveal Ecd as a novel marker for breast cancer progression and show that levels of Ecd expression predict poorer survival in Her2/neu overexpressing breast cancer patients.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-011-1946-8) contains supplementary material, which is available to authorized users.
PMCID: PMC3397230  PMID: 22270930
Ecdysoneless; Cell cycle; Breast cancer; HER2/neu; DCIS; IDC
24.  Prognostic value of proliferation assay in the luminal, HER2-positive, and triple-negative biologic classes of breast cancer 
Although the prognostic significance of proliferation in early invasive breast cancer has been recognized for a long time, recent gene-expression profiling studies have reemphasized its biologic and prognostic value and the potential application of its assessment in routine practice, particularly to define prognostic subgroups of luminal/hormone receptor-positive (HR+) tumors. This study aimed to assess the prognostic value of a proliferation assay by using Ki-67 immunohistochemistry as compared with mitotic count scores.
Proliferation was assessed by using Ki-67 labeling index (Ki-67LI) and mitotic scores in a large (n = 1,550) and well-characterized series of clinically annotated primary operable invasive breast cancer with long-term follow-up. Tumors were phenotyped based on their IHC profiles into luminal/HR+, HER2+, and triple-negative (TN) classes. We used a split-sample development and validation approach to determine the optimal Ki-67LI cut-offs.
The optimal cut-points of Ki-67LI were 10% and 50% for the luminal class. Both Ki7LI and MS were able to split luminal tumors into subgroups with significantly variable outcomes, independent of other variables. Neither mitotic count scores nor Ki-67LI was associated with outcome in the HER2+ or the TN classes.
Assessment of proliferation by using Ki-67LI and MS can distinguish subgroups of patients within luminal/hormone receptor-positive breast cancer significantly different in clinical outcomes. Overall, both Ki-67 LI and mitotic-count scores showed comparable results. The method described could provide a cost-effective method for prognostic subclassification of luminal/hormone receptor-positive breast cancer in routine clinical practice.
PMCID: PMC3496118  PMID: 22225836
25.  The number of women who would need to be screened regularly by mammography to prevent one death from breast cancer 
Journal of Medical Screening  2011;18(4):210-212.
The number of women who would need to be screened regularly by mammography to prevent one death from breast cancer depends strongly on several factors, including the age at which regular screening starts, the period over which it continues, and the duration of follow-up after screening. Furthermore, more women would need to be INVITED for screening than would need to be SCREENED to prevent one death, since not all women invited attend for screening or are screened regularly. Failure to consider these important factors accounts for many of the major discrepancies between different published estimates. The randomised evidence indicates that, in high income countries, around one breast cancer death would be prevented in the long term for every 400 women aged 50–70 years regularly screened over a ten-year period.
PMCID: PMC3266234  PMID: 22184734

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