Sary This study aimed to determine the expession of Nm23-H1 in colorectal cancer and lver metastases and to correlate N1m23-HI expression with dinicopathological variabe Simens from 59 primary colorctal camns and five liver metastases were studied usin Northern blot hybriTisauion. The mean ± sc. of tumour/normal (I/N) ratio of Nm23-H1 RNA expreson was 4.3 ± 0.4 (P<0.001) and 5.1 ± 0.90 (P<0.01) for co_oectal cancer and iver tsases rI ty. No significant relationship was observed b n the klv of Nm23-Hl RNA and the patent's age, scx, tumour location, differntiation, prmnce of lymph node involvement or distant metatases Nm23-H1 RNA kvel was 2.6 ± 0.5 for tumour size less than 3.0 an and 4.6 ± 0.5 for those > 3.0 an (P = 0.05). Ther a ed to be a trend between ireasg lative Nm23-Hl RNA and bowel wal invasi, ispec of metastatic status (Ti = 1.9 ± 0.3, T2 = 4.1 0.6, T3 = 4.1 ± 0.5 and T4 = 6.4 ± 1.6). Tumour specimens were obtained from the tumour edge, thus avoiding a necrotic centre. Gross normal
SummaryN (7-carboxyheptyl) imidazole is an inhibitor of platelet thromboxane synthetase that has no effect on the cyclooxygenase activity. An oral dose of the substance to rats (10 mg/kg) prolonged tail bleeding time from 170 ± 13 sec to 284 ± 22 sec. This oral dose also inhibited platelet thromboxane B2 production induced by collagen ex vivo but had little effect on the aggregation dose response curve. There was no effect on thrombin-induced aggregation.Neither the thrombocytopenia induced by the Arthus reaction nor thrombus formation on an implanted cotton thread were inhibited by oral doses of carboxyheptylimidazole up to 30 mg/kg. Similarly neither the prothrombin nor activated partial thromboplastin time were affected.It is postulated that this thromboxane synthetase inhibitor prolongs bleeding time not by inhibiting platelet aggregation or blood coagulation but rather by preventing the vasoconstriction which would normally be caused by thromboxane A2.
Introduction: IL6ST is regarded as a putative ER target gene. Recently it has been recognised as a key biomarker for prediction of response to endocrine therapy (ET), having been included as the primary biomarker in our EA2Clin test and as an ER-signalling gene in the EndoPredict test. In both tests higher IL6ST expression is associated with a better response to ET and better prognosis. Despite its importance as a biomarker, little is known about its functional role in breast cancer (BC). Methods: Pre- and on-treatment (at 14-days and at surgery) samples were collected from 102 post-menopausal women with ER+ BC, treated with 3-6 months of neoadjuvant ET. RNA was extracted for whole-genome expression analysis. From a subset with available fresh frozen tissue (28 patients, 83 samples) protein was extracted and proteome analysis using mass spectrometry is currently underway – results available for SABCS 2017. Immunohistochemistry was performed on FFPE tissue microarrays (TMAs) comprising pre-treatment samples from 102 patients. Cytoplasmic/membrane staining was scored using a graduated scale (0-3+) and nuclear staining was graded using an Immunoscore. Results: IL6ST exists in membrane-bound and soluble forms of varying size. The full-length membrane bound molecule comprises 8 domains: 6 extracellular, 1 transmembrane and 1 cytoplasmic. In the EA2Clin test, pre-treatment BC tissues are stained for IL6ST with an antibody specific for a region spanning the transmembrane and cytoplasmic domains. TMAs were stained for IL6ST with both this and a second antibody binding the extracellular part, detecting both full-length and most soluble isoforms. Levels of both were correlated (R=0.82, P<0.0001). IL6ST is known to mediate the action of cytokines including IL6, OSM and LIF via downstream regulation of pathways such as JAK/STAT. TMAs were stained for antibodies against IL6ST, OSM, IL6, total STAT3, pSTAT3 (Tyr705) and pSTAT3 (Ser727). IL6ST was scored as low (0/1+) or high (2+/3+). There was a positive association between levels of IL6ST and IL6 (P=0.02) and total STAT3 (P=0.003). There was no association between IL6ST and OSM or either pSTAT3. Supervised gene expression analysis comparing pre-treatment samples with high and low IL6ST levels revealed increased levels of STAT3-regulated genes: cell cycle (CEBPD, CDKN1B), apoptosis (NFIL3, ATF3, BCL2), extracellular matrix remodelling (ADM, SEPRINE1-3) and interferon signalling (IFIT1, IFI44, IFI27). Unsupervised gene enrichment analysis revealed increased expression of genes involved with JAK/STAT, PI3K, mTOR and ERBB1 signalling in tumours expressing higher IL6ST levels. Lower levels were associated with increased energy generation, cellular metabolism and epithelial-mesenchymal transition. Conclusions: • This is the first matched whole-genome and mass spectrometry proteome analysis of sequential ET-treated BC patients • IL6ST predicts response to ET – it is used in2 independent assays • Levels of full-length IL6ST appear to be the most important for ET response prediction • IL6ST may have an active role in BC cells, mediating signalling of cytokines such as IL6 through the JAK/STAT pathway and subsequent downstream transcriptional regulation. Citation Format: Turnbull AK, Fernando A, Martinez-Perez C, Finch AJ, von Kriegsheim A, Wills J, Quinn N, Selli C, Mosley D, Langdon SP, Sims AH, Dixon JM. Understanding the mechanisms of action underlying the role of IL6ST, a key biomarker for prediction of response to endocrine therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-08-02.
Background Interest in the ubiquitously expressed transmembrane receptor IL6ST (gp130) has developed as it has been identified as a predictive biomarker of endocrine treatment response in breast cancer patients and is included in the 'Endopredict' test. At least seven cytokines (IL-6, OSM, LIF, IL-11, CNTF, IL-27, CT-1) signal via IL6ST. Interleukin-6 (IL-6) can mediate effects via two signaling pathways; classic signaling (through the membrane-bound IL-6 receptor, IL-6R) and trans-signaling (via non-signaling membrane-bound soluble IL-6R, sIL-6R). Both pathways may occur in parallel and activate cells. Crosstalk between the ER signaling pathway and IL6ST through STAT3 has been identified and implicated in the conversion from ER responsiveness to non-responsiveness. Method A panel of 3 ERa+ luminal breast cancer cell lines (MCF-7, T47D, ZR-75-1) were chosen to examine IL6ST expression by western blot, gel electrophoresis and qRT-PCR. Proliferation assays (SRB) were carried out to investigate the effects of seven cytokines (IL-6, OSM, LIF, IL-11, CNTF, IL-27, CT-1) on cell growth. The action of both IL-6 and Oncostatin M (OSM) on cell migration and downstream signaling pathways (pSTAT3 and pERK1/2) was studied. The extent of trans-signaling occurrence and interaction between cytokines and estrogen was investigated using proliferation assays. Results Three cell lines (MCF-7, T47D, ZR-75-1 were shown to express varying levels of full-length IL6ST, with MCF-7 having the least expression. Gel electrophoresis and qRT-PCR confirmed the presence of all previously described IL6ST soluble forms in the three cell lines. Surprisingly, the growth response to the cytokines was variable across the cell lines. IL6 caused a modest increase in growth in MCF-7 but produced inhibition in ZR-75-1. OSM and LIF stimulated growth in MCF-7, whereas OSM inhibited ZR-75-1 and LIF had no effect. Interestingly, no significant effect on growth was seen in T47D with any of the cytokines except IL-11 which generated a significant growth effect on T47D cells. Both STAT3 and MAPK/ERK pathways were activated to different extents in the three cell lines as a result of OSM and IL-6 activation, whereas cells migration was only stimulated in ZR-75-1. The trans-signaling pathway significantly enhanced the growth inhibition in ZR-75-1. Estrogen eliminated the effect of both IL-6 and OSM on MCF-7 growth, while both cytokines decreased estrogen-induced cell proliferation in ZR-75-1. There was no effect in T47D. Conclusion These results indicate: · The presence of both classic and trans-signaling occurrence in breast cancer cell lines. · Differential patterns of pSTAT3 and pERK1/2 signaling which could help explain the variation in responses to the cytokines. · IL6ST full-length form is the most abundant form in all three cell lines under basal conditions. · The levels and roles of the different IL6ST soluble forms will be further studied after estrogen stimulation. · The effect of IL6ST silencing in the presence of estrogen and tamoxifen on cell growth and the gene expression currently being examined. Citation Format: Mosly DH, Turnbull AK, Langdon SP, Sims AH. A potential role for IL6ST mediating endocrine resistance in breast cancer via interaction with the ER signaling pathway [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-28.
Background: ER+/HER2+ accounts for up to 10% of all breast cancers (BCs) and most are treated with endocrine therapy (ET) after surgery to reduce the recurrence risk. We developed and validated an immunohistochemistry (IHC) based test (EA2Clin) that incorporates baseline IL6ST, clinical variables and on-treatment measurement of MCM4. Responders (Rs) and non-responders (NRs) to ET are identified and it accurately estimates recurrence-free survival (RFS) and BC-specific overall survival (BCSS). The aim was to determine if EA2Clin could accurately predict ER+/HER2+ patients likely to benefit from ET and to determine if it can identify those for whom HER2-targeted therapies are required. Methods: 3 cohorts were studied: A: 32 post-menopausal women (PMW) with large ER+/HER2+ BC treated with neoadjuvant (3-6 months) then adjuvant letrozole. 5 also received adjuvant chemotherapy plus Herceptin. Neoadjuvant clinical response was assessed by changes in tumour volume. Tumour core biopsies were taken at 0, 14 days and 3 months. Gene expression analysis using Illumina HT12 whole-genome beadarrays was performed on a subset (n=17) where fresh tissue was available. B: 13 PMW with ER+/HER2+ BC who were treated by surgery without neoadjuvant therapy. RNA was extracted from excision tissues and analysed using whole-genome Affymetrix U133A microarrays. C: 15 PMW with ER+/HER2+ BC treated with 2-weeks of pre-operative letrozole (n=7) or anastrozole (n=8). All received adjuvant letrozole. Tissues were collected at pre-treatment and at surgery. None received Herceptin or chemotherapy. All patients were followed-up after surgery (median follow-up = 6.4 years). Results: In cohort A, half (16/32) of the patients responded to ET with tumour volume reductions of >70% with neoadjuvant treatment. Innate resistance was apparent in 3 patients with continued tumour growth on ET, whereas 13 patients developed resistance after a period of response. EAClin2 predicted neoadjuvant response with a 92% accuracy. There was increased expression of phospho-AKT and phospho-ERK in NRs, not seen in Rs. Half (8/16) of the NR cancers expressed phospho-ER; but was not seen in any responsive cancer. Gene expression analysis in 17 patients showed increased MAPK and PI3K pathway activity in the 9 NR compared with the 8 R tumours. These results were recapitulated in cohort B where MAPK and PI3K activity were associated with low levels of IL6ST. In the 16/32 patients who responded well to neoadjuvant ET the actuarial recurrence rate was 0% at 5 and 10 years. The rate of recurrence in the NR was 30% at both 5 and 10 years. Of the 5 patients who received chemotherapy plus Herceptin, none recurred despite a poor response to neoadjuvant letrozole (median length to last follow-up was 6.1 years). Initial data suggest that in cohort B EA2Clin identifies a group of ER+/HER2+ cancers that can be managed by ET alone. Conclusions: · The EA2Clin test identifies ER+/HER2+ BCs who respond well to ET alone and those with a poor clinical response who have higher risk of recurrence. · NR to ET have increased expression of PI3K and MAPK pathways, consistent with active HER2 signalling. · There is potential role for EA2Clin in selecting ER+/HER2+ patients that require and benefit from HER2-targeted therapies. Citation Format: Turnbull AK, Webber V, McStay D, Arthur L, Martinez-Perez C, Fernando A, Renshaw L, Keys J, Clarke R, Sims AH, Dixon JM. Predicting benefit from HER2-targeted therapies in patients with ER+/HER2+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-26.
The role of thromboxane A2 (TXA2) in haemostasis was investigated with the use of a selective inhibitor of platelet thromboxane synthetase used in conjunction with radioimmunoassay of thromboxane B2 (TXB2). N-Carboxyheptylimidazole is such an inhibitor having no effect on platelet cyclooxygenase. An oral dose of this substance (10 mg/kg) to rats resulted in 85% (P < 0.001) suppression of platelet TXB2 production induced by collagen ex vivo while the ED50 and maximum rate of platelet aggregation were unchanged. It also caused a prolongation of tail bleeding time from 153±13 to 284±22 secs (P < 0.01). The thrombocytopenia resulting from the Arthus reaction in rats was unchanged, and the prothrombin and activated partial thromboplastin coagulation times were not affected by either 10 or 30 mg/kg p.o. It is concluded that the role of TXA2 in prevention of rat tail bleeding is not as an activator of platelet aggregation or blood coagulation. It is more likely that TXA2 prevents bleeding via its potent vaso-constricting properties. In addition the increased bleeding time may be due to change in the equilibrium of other vasoactive prostanoids.
Purpose: Neo-Adjuvant chemotherapy treatment is increasingly being used in breast cancer to preoperatively shrink tumour volumes and facilitate surgical plans. These datasets however, are still scarce, making it difficult to assess the relative value of multiple time point biopsies compared to diagnostic only sampling. This study aims to identify sequential samplings intrinsic value. Method: A total of 97 samples from a cohort of 50 neoadjuvant chemotherapy treated primary breast cancer patients (aged 29-76 at diagnosis, Allred status 47:53% +/-, Her2 status 80:20% +/-, mixed grade and menopausal status) taken pre- treatment, at 2 weeks on-treatment, mid chemotherapy and at resection were sequenced with Ion Ampliseq transcriptome yielding expression values for 12,635 genes. Differential expression analysis was performed across response groups (16 Responders, 34 Non-Responders) as defined by Pathological Complete Response and over treatment time to identify significantly differentially expressed genes, pathways and markers indicative of response status. Results: An on-treatment marker for response was identified (AAGAB), which resulted in a testing accuracy of 100% and a validation accuracy of 78% in the I-SPY 1 Trial. AAGAB was predictive of long term survival (p = 0.048 testing, p = 0.031 validation) in both chemotherapy cohorts at the same expression level as defined for treatment response. The single gene on-treatment biomarker, AAGAB proves more performant than established prognostic tests, Mammaprint (Edinburgh NEO trial, pre-treatment 61%, on-treatment 63%. I-SPY 1 trial, pre-treatment 60%, on-treatment 66%) and Pam50 RORS (neo trial pre-treatment 50%, on treatment 58%, Magbanua trial pre-treatment 56%, on-treatment 64%) Conclusion: Changes in gene expression of on-treatment chemotherapy breast cancer resulted in the identification of a novel gene marker that was as effective in predicting prognostic status as established prognostic tests. These results support the use of on-treatment testing in breast cancer to improve the accuracy of tumour response prediction. Citation Format: Bownes RJ, Turnbull AK, Cameron D, Sims AH, Oikonomidou O. On-treatment biomarkers can improve prediction of response to neoadjuvant chemotherapy in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-11-13.
Background: The majority of patients with early-stage estrogen receptor positive (ER+) breast cancer (BC) are treated with adjuvant endocrine therapy (ET) after primary surgery to reduce the risk of recurrence. A variety of tests are available to predict outcomes on ET but most require gene-level measurements and are expensive. Recently, we developed an immunohistochemistry (IHC) based test (EA2Clin) using levels of pre-treatment IL6ST together with clinical variables and on-treatment proliferation. The aim was to validate this test in cohorts of both pre- and post-menopausal women treated with two weeks of a variety of endocrine treatments (tamoxifen, fulvestrant or an aromatase inhibitor) prior to surgery. Methods: The cohorts are: (A) 186 post-menopausal women (PMW) with ER+ BC treated with at least 2 weeks of preoperative or neoadjuvant letrozole or anastrozole, then surgery followed by adjuvant letrozole (n=132) or tamoxifen (n=54); (B) 51 pre-menopausal women (preMW) with ER+ BC treated with 2 weeks of either neoadjuvant tamoxifen (n=24) or one 750mg dose of faslodex (n=27), then surgery followed by adjuvant tamoxifen. The median follow-up was 5.4 years for cohort A and 10.2 years for cohort B. IHC analysis was performed using a Leica BOND III autostainer and the EA2Clin algorithm was used to stratify patients in binary high or low-risk groups. Results: In the cohort of PMW, EA2Clin was highly significantly associated with both recurrence-free survival (RFS) (P<0.0001, HR=13.26, 95%CI=5.59-13.46) and breast cancer specific survival (BCSS) (P<0.0001, HR=12.93, 95%CI=4.43-37.72). The 5 and 10 year actuarial recurrence rates were 7%/22% and 46%/73% for the low and high risk groups, respectively. The actuarial breast cancer-related death rate for the low risk group was 5% at both 5 and 10 years, whereas for the high risk group was 33%/38%. Confounding factors were not found to be significant. In the cohort of preMW, our test was significantly associated with both RFS (P=0.002, HR=5.71, 95%CI=1.91-17.05) and BCSS (P=0.016, HR=4.81, 95%CI=1.34-17.26). The 5 and 10 year actuarial recurrence rates were 12%/29% and 27%/77% for the low and high risk groups, respectively. The 5 and 10 year actuarial breast cancer-related death rates were 7%/19% and 9%/58% for low and high risk groups, respectively. Discussion: · This study has validated EA2Clin as the first predictive tool to incorporate clinical data with pre and on-treatment immunohistochemical biomarkers to predict accurately the outcome of patients with ER positive breast cancer treated with adjuvant ET. · This test predicts both RFS and BCSS in pre- and PMW treated with a variety of endocrine agents. · Because this test incorporates clinical variables with simple IHC, it can be performed locally in any pathology lab. Citation Format: Turnbull AK, Fernando A, Renshaw L, Keys J, Thomas JS, Sims AH, Dixon JM. EA2Clin: A novel immunohistochemical prognostic and predictive test for patients with estrogen receptor-Positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-08-03.
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