The role of infiltrating lymphocytes in the neo-adjuvant treatment of women with HER2-positive breast cancer.

BACKGROUND: Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response (pCR) in HER2-positive breast cancer. Whilst studies have correlated baseline lymphocyte levels with subsequent pCR, few have studied the impact of neoadjuvant therapy on the immune environment. METHODS: We performed TIL analysis and T-cell analysis by IHC on the pretreatment and 'On-treatment' samples from patients recruited on the Phase-II TCHL (NCT01485926) clinical trial. Data were analysed using the Wilcoxon signed-rank test and the Spearman rank correlation. RESULTS: In our sample cohort (n = 66), patients who achieved a pCR at surgery, post-chemotherapy, had significantly higher counts of TILs (p = 0.05) but not SLs (p = 0.08) in their pre-treatment tumour samples. Patients who achieved a subsequent pCR after completing neo-adjuvant chemotherapy had significantly higher SLs (p = 9.09 × 10-3) but not TILs (p = 0.1) in their 'On-treatment' tumour biopsies. In a small cohort of samples (n = 16), infiltrating lymphocyte counts increased after 1 cycle of neo-adjuvant chemotherapy only in those tumours of patients who did not achieve a subsequent pCR. Finally, reduced CD3 + (p = 0.04, rho = 0.60) and CD4 + (p = 0.01, rho = 0.72) T-cell counts in 'On-treatment' biopsies were associated with decreased residual tumour content post-1 cycle of treatment; the latter being significantly associated with increased likelihood of subsequent pCR (p < 0.01). CONCLUSIONS: The immune system may be 'primed' prior to neoadjuvant treatment in those patients who subsequently achieve a pCR. In those patients who achieve a pCR, their immune response may return to baseline after only 1 cycle of treatment. However, in those who did not achieve a pCR, neo-adjuvant treatment may stimulate lymphocyte influx into the tumour.

Indexing electron backscatter diffraction patterns with a refined template matching approach.

Electron backscatter diffraction (EBSD) is a well-established method of characterisation for crystalline materials. Using this technique, we can rapidly acquire and index diffraction patterns to provide phase and orientation information about the crystals on the material surface. The conventional analysis method uses signal processing based on a Hough/Radon transform to index each diffraction pattern. This method is limited to the analysis of simple geometric features and ignores subtle characteristics of diffraction patterns, such as variations in relative band intensities. A second method, developed to address the shortcomings of the Hough/Radon transform, is based on template matching of a test experimental pattern with a large library of potential patterns. In the present work, the template matching approach has been refined with a new cross correlation function that allows for a smaller library and enables a dramatic speed up in pattern indexing. Refinement of the indexed orientation is performed with a follow-up step to allow for small alterations to the best match from the library search. The refined template matching approach is shown to be comparable in accuracy, precision and sensitivity to the Hough based method, even exceeding it in some cases, via the use of simulations and experimental data collected from a silicon single crystal and a deformed α-iron sample. The speed up and pattern refinement approaches should increase the widespread utility of pattern matching approaches.

Derivation of the clinical grade human embryonic stem cell line RCe021-A (RC-17).

The human embryonic stem cell line RCe021-A (RC-17) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a day 3 embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe021-A (RC-17) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the clinical grade human embryonic stem cell line RCe013-A (RC-9).

The human embryonic stem cell line RCe013-A (RC-9) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following informed consent. RCe013-A (RC-9) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the clinical grade human embryonic stem cell line RCe015-A (RC-11).

The human embryonic stem cell line RCe015-A (RC-11) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a fragmented cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe015-A (RC-11) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the human embryonic stem cell line RCe009-A (RC-5).

The human embryonic stem cell line RCe009-A (RC-5) was derived from a frozen and thawed Day 2 embryo voluntarily donated as unsuitable and surplus to requirement for fertility treatment following informed consent under licence from the UK Human Fertilisation and Embryology Authority. RCe009-A carries the common DF508 mutation on the cystic fibrosis trans-membrane regulator gene associated with the disease cystic fibrosis. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the human embryonic stem cell line RCM1.

The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Derivation of the human embryonic stem cell line RCe010-A (RC-6).

The human embryonic stem cell line RCe010-A (RC-6) was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the human embryonic stem cell line RCe011-A (RC-7).

The human embryonic stem cell line RCe011-A (RC-7) was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the human embryonic stem cell line RCe012-A (RC-8).

The human embryonic stem cell line RCe012-A (RC-8) was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Derivation of the human embryonic stem cell line RCe014-A (RC-10).

The human embryonic stem cell line RCe014-A (RC-10) was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Derivation of the clinical grade human embryonic stem cell line RCe019-A (RC-15).

The human embryonic stem cell line RCe019-A (RC-15) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe019-A (RC-15) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XX/47XX, +8 female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Derivation of the clinical grade human embryonic stem cell line RCe017-A (RC-13).

The human embryonic stem cell line RCe017-A (RC-13) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a frozen and thawed blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe017-A (RC-13) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 47XY, +12/48XY, +1, +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the clinical grade human embryonic stem cell line RCe018-A (RC-14).

The human embryonic stem cell line RCe018-A (RC-14) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe018-A (RC-14) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a male karyotype with an extra copy of chromosome 8 (47XY, +8). Microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12).

The human embryonic stem cell line RCe016-A (RC-12) was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP) standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12) shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15), which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available.

Derivation of the clinical grade human embryonic stem cell line RCe020-a (RC-16).

The human embryonic stem cell line RCe020-A (RC-16) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe020-A (RC-16) shows normal pluripotency marker expression and differentiates to mesoderm and potentially ectoderm in vitro. It has an abnormal 47XX, +14, i(20)(q10) female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Kinase inhibitor screening identifies CDK4 as a potential therapeutic target for melanoma.

Despite recent advances in targeted therapies and immunotherapies metastatic melanoma remains only rarely curable. The objective of the present study was to identify novel therapeutic targets for metastatic melanoma. A library of 160 well-characterised and potent protein kinase inhibitors was screened in the BRAF mutant cell line Sk-Mel-28, and the NRAS mutant Sk-Mel-2, using proliferation assays. Of the 160 inhibitors tested, 20 achieved >50% growth inhibition in both cell lines. Six of the 20 were cyclin dependent kinase (CDK) inhibitors, including two CDK4 inhibitors. Fascaplysin, a synthetic CDK4 inhibitor, was further tested in 8 melanoma cell lines. The concentration of fascaplysin required to inhibit growth by 50% (IC50 value) ranged from 0.03 to 0.22 µM. Fascaplysin also inhibited clonogenic growth and induced apoptosis. Sensitivity to PD0332991, a therapeutic CDK4/6 inhibitor was also evaluated in the melanoma cell lines. PD0332991 IC50 values ranged from 0.13 to 2.29 µM. Similar to fascaplysin, PD0332991 inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells in vitro, suggesting that CDK4 may be a rational therapeutic target for metastatic melanoma.

Horse impoundments under Control of Horses legislation in the Munster region of Ireland: factors affecting euthanasia.

Recently, considerable international attention has been paid to the problem of unwanted horses. In Ireland, stray horses, particularly in urban areas, are a further problem. The Control of Horses Act 1996 was enacted in response to an ongoing problem of uncontrolled horses in public places. As yet, no research work has been conducted focusing on stray horses in Ireland. This paper describes horses impounded under the Act in the Munster region of Ireland during 2005-2012 and the factors influencing decisions regarding their disposal. A logistic regression model was developed to investigate factors influencing the probability that a horse was euthanised during impoundment. In total, 3625 seizure events were recorded, most towards the end of the study period. Predictors for euthanasia during 2010-2012 included seizure location, sex, age, colour, body condition score and year. This study highlights the problem of stray horses in Ireland, particularly in urban areas. There is a need for rigorous enforcement of newly enacted horse identification legislation, allowing a fully integrated traceability system. More is required to manage the long-established societal problems of stray horses in urban settings, with a uniform approach by all Local Authorities being long overdue.

HER2-family signalling mechanisms, clinical implications and targeting in breast cancer.

Approximately 20 % of human breast cancers (BC) overexpress HER2 protein, and HER2-positivity is associated with a worse prognosis. Although HER2-targeted therapies have significantly improved outcomes for HER2-positive BC patients, resistance to trastuzumab-based therapy remains a clinical problem. In order to better understand resistance to HER2-targeted therapies in HER2-positive BC, it is necessary to examine HER family signalling as a whole. An extensive literature search was carried out to critically assess the current knowledge of HER family signalling in HER2-positive BC and response to HER2-targeted therapy. Known mechanisms of trastuzumab resistance include reduced receptor-antibody binding (MUC4, p95HER2), increased signalling through alternative HER family receptor tyrosine kinases (RTK), altered intracellular signalling involving loss of PTEN, reduced p27kip1, or increased PI3K/AKT activity and altered signalling via non-HER family RTKs such as IGF1R. Emerging strategies to circumvent resistance to HER2-targeted therapies in HER2-positive BC include co-targeting HER2/PI3K, pan-HER family inhibition, and novel therapies such as T-DM1. There is evidence that immunity plays a key role in the efficacy of HER-targeted therapy, and efforts are being made to exploit the immune system in order to improve the efficacy of current anti-HER therapies. With our rapidly expanding understanding of HER2 signalling mechanisms along with the repertoire of HER family and other targeted therapies, it is likely that the near future holds further dramatic improvements to the prognosis of women with HER2-positive BC.