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Breast cancer is the most common malignancy in women, with the majority classified as hormone receptor–positive (HR+) and HER2 receptor–negative (HER2−). After the approval of the CDK4/6 inhibitors, palbociclib, ribociclib and abemaciclib for advanced HR+/HER2− breast cancer, combinations of endocrine therapy (ET) and CDK4/6 inhibitors are becoming routine practice. However, drug resistance remains an important issue. To overcome this, multiple targeted therapies are in development, and several approaches are in clinical trial in combination with ET, including drugs that inhibit CDK7, FGFR, AURKA, HDAC and the PI3K–AKT–mTOR pathway. Triple combinations of investigational drugs with ET and CDK4/6 inhibitors are also being trialled.

Background: The effect of the COVID-19 on breast cancer treatment is unknown. The pandemic’s impact on treatment delivery was assessed in patients undergoing chemotherapy treatment at a major London oncology centre. Methods: Treatment and medical records for all patients attending the chemotherapy unit and on outpatient treatment for breast cancer over an 8 week period (during first lockdown; 23rd March-17th May 2020) were compared to a similar time period in 2019. Results: Breast cancer diagnosis referrals fell by 38% mainly due to screening services halting which had a knock-on effect on patient numbers starting their first treatment (reduced by 34%). Neoadjuvant, adjuvant, and palliative patients were all affected, mostly at the start of lockdown. On-site chemotherapy and supportive treatments fell by 39% compared to 2019, mainly due to fewer number of bone-modifying agents. Towards the end of 8 week period, treatment numbers had nearly returned to normal. The system adapted by modifying treatment regimens, using telemedicine, increased use of supportive medications and less frequent blood tests. Conclusion: COVID-19’s global impact has significantly reduced breast cancer treatments given during the first lockdown. Whilst recovery is now evident, cancer services, patients, and clinical cancer research must be prioritised in future waves.

Neutrophils (Ns) influence cancer cell survival and metastasis through the release of chemokines, cytokines and interactions within the microenvironment and at distal sites from the primary tumour. Background: Patients with Breast Cancer (BC) often display elevated levels of circulating blood neutrophils and an elevated neutrophil: lymphocyte ratio (NLR) is associated with a shorter disease-free survival in patients with Early Breast Cancer (EBC). Evidence from mouse models of BC shows that Ns can either have a tumour-promoting or tumour-inhibitory role. CD62L (L-selectin) is a cell adhesion molecule found on the surface of leukocytes which is important for helping circulatory Ns bind to the endothelium and migrate into tissues and is rapidly shed upon N activation. Our laboratory has found a lower proportion of Ns expressing CD62L in pre-clinical models of BC, which implies that Ns from tumour-bearing mice have less endothelial- adhesive capacity and hence will remain in the circulation for a longer time. However, there is limited knowledge regarding whether different N subpopulations are present within the circulation of patients with BC, how early this occurs in the disease process and whether this is influenced by BC phenotype. In order to address these questions, we phenotyped Ns from BC patients. Objectives: Our primary objectives were to investigate changes in the surface marker CD62L expression via flow cytometry in neutrophils from patients and HVs to see if the changes seen in the pre-clinical mouse models were also seen in patients. Since kinases govern many aspects of intracellular signalling and functions, we also assessed the Serine Threonine Kinase (STK) and Phosphotyrosine Kinase (PK) activity within circulatory neutrophils in patients with Breast Cancer and paired HVs using a Pamgene kinase assay. This evaluated the activity of kinases (n = 340 kinases) in N lysates to compare N kinase activity in EBC patients and age-matched HVs. Methods: We recruited 28 patients with Early BC (EBC) (10 Node +, 18 node -; 10 T1 and 16 T2 and 2 T3) and paired healthy volunteers (HVs) who were age and sex-matched to compare phenotypical features of Ns isolated from the blood. 21 Patients with Oestrogen receptor positive (ER+) and 7 with Oestrogen receptor negative (ER -) EBC were recruited between June 2018 and June 2021. No patient had overt metastatic disease and all were screened for exclusion factors, such as infection, autoimmune conditions and certain medication known to influence neutrophils. Results: We found that ER+ patients had a lower level of the CD62Llow neutrophil subset compared to HVs (P< 0.0375). In contrast there was a significant increase in the CD62Llow neutrophil subset (2-fold higher) for ER - patients compared to HVs (P< 0.0316).The Pamgene kinase assay showed that there was a general upregulation in both STK and PK kinase activity, especially the tyrosine family of kinases for patients with ER positive breast cancer (Median kinase score which is the combined sensitivity score and specificity score > 1.2) and a general downregulation in kinase activity, especially the CMGC family of kinases for patients with ER negative disease (Median kinase score > 1.2) compared to paired HVs. The functional implications of these differences in kinase activity are being actively investigated. Conclusion: We have detected changes in CD62L expression and neutrophil kinase activity within the circulation of patients with EBC and the nature of these changes appear to be intrinsically linked to ER subtype. These findings may have important implications for use as part of an early diagnostic multi-omic platform. Citation Format: Anisha Ramessur, Bana Ambasager, Ilaria Malanchi, Raoul Charles Coombes. The multi switching activity of neutrophils in the circulation of patients with early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-14.

The peri‐operative use of angiotensin‐converting enzyme inhibitors or angiotensin‐2 receptor blockers is thought to be associated with an increased risk of postoperative acute kidney injury. To reduce this risk, these agents are commonly withheld during the peri‐operative period. This study aimed to investigate if withholding angiotensin‐converting enzyme inhibitors or angiotensin‐2 receptor blockers peri‐operatively reduces the risk of acute kidney injury following major non‐cardiac surgery. Patients undergoing elective major surgery on the gastrointestinal tract and/or the liver were eligible for inclusion in this prospective study. The primary outcome was the development of acute kidney injury within seven days of operation. Adjusted multi‐level models were used to account for centre‐level effects and propensity score matching was used to reduce the effects of selection bias between treatment groups. A total of 949 patients were included from 160 centres across the UK and Republic of Ireland. From this population, 573 (60.4%) patients had their angiotensin‐converting enzyme inhibitors or angiotensin‐2 receptor blockers withheld during the peri‐operative period. One hundred and seventy‐five (18.4%) patients developed acute kidney injury; there was no difference in the incidence of acute kidney injury between patients who had their angiotensin‐converting enzyme inhibitors or angiotensin‐2 receptor blockers continued or withheld (107 (18.7%) vs. 68 (18.1%), respectively; p = 0.914). Following propensity matching, withholding angiotensin‐converting enzyme inhibitors or angiotensin‐2 receptor blockers did not demonstrate a protective effect against the development of postoperative acute kidney injury (OR (95%CI) 0.89 (0.58–1.34); p = 0.567).

The role of estrogen receptor (ER) α as a target in treatment of breast cancer is clear, but those of ERβ1 and ERβ2 in the breast remain unclear. We have examined expression of all three receptors in surgically excised breast samples from two archives: (i): 187 invasive ductal breast cancer from a Japanese study; and (ii) 20 lobular and 24 ductal cancers from the Imperial College. Samples contained normal areas, areas of hyperplasia, and in situ and invasive cancer. In the normal areas, ERα was expressed in not more than 10% of epithelium, whereas approximately 80% of epithelial cells expressed ERβ. We found that whereas ductal cancer is a highly proliferative, ERα-positive, ERβ-negative disease, lobular cancer expresses both ERα and ERβ but with very few Ki67-positive cells. ERβ2 was expressed in 32% of the ductal cancers, of which 83% were postmenopausal. In all ERβ2-positive cancers the interductal space was filled with dense collagen, and cell nuclei expressed hypoxia-inducible factor 1α. ERβ2 expression was not confined to malignant cells but was strong in stromal, immune, and endothelial cells. In most of the high-grade invasive ductal cancers neither ERα nor ERβ was expressed, but in the high-grade lobular cancer ERβ was lost and ERα and Ki67 expression were abundant. The data show a clear difference in ER expression between lobular and ductal breast cancer and suggest (i) that tamoxifen may be more effective in late than in early lobular cancer and (ii) a potential role for ERβ agonists in preventing in situ ductal cancers from becoming invasive.

Background Randomized trials of radiotherapy and surgery for early breast cancer may have been too small to detect differences in long-term survival and recurrence reliably. We therefore performed a systematic overview (meta-analysis) of the results of such trials. Methods Information was sought on each subject from investigators who conducted trials that began before 1985 and that compared local therapies for early breast cancer. Data on mortality were available from 36 trials comparing radiotherapy plus surgery with the same type of surgery alone, 10 comparing more-extensive surgery with less-extensive surgery, and 18 comparing more-extensive surgery with less-extensive surgery plus radiotherapy. Information on mortality was available for 28,405 women (97.4 percent of the 29,175 women in the trials). Results The addition of radiotherapy to surgery resulted in a rate of local recurrence that was three times lower than the rate with surgery alone, but there was no significant difference in 10-year survival; among a total of 17,273 women enrolled in such trials, mortality was 40.3 percent with radiotherapy and 41.4 percent without radiotherapy (P = 0.3). Radiotherapy was associated with a reduced risk of death due to breast cancer (odds ratio, 0.94; 95 percent confidence interval, 0.88 to 1.00; P = 0.03), which indicates that, after 10 years, there would be about 0 to 5 fewer deaths due to breast cancer per 100 women. However, there was an increased risk of death from other causes (odds ratio, 1.24; 95 percent confidence interval, 1.09 to 1.42; P = 0.002). This, together with the age-specific death rates, implies, after 10 years, a few extra deaths not due to breast cancer per 100 older women or per 1000 younger women. During the first decade or two after diagnosis, the excess in the rate of such deaths that was associated with radiotherapy was much greater among women who were over 60 years of age at randomization (15.3 percent vs. 11.1 percent [339 vs. 249 deaths]) than among those under 50 (2.5 percent vs. 2.0 percent [62 vs. 49 deaths]). Breast-conserving surgery involved some risk of recurrence in the remaining tissue, but no significant differences in overall survival at 10 years were found in the studies of mastectomy versus breast-conserving surgery plus radiotherapy (4891 women), more-extensive surgery versus less-extensive surgery (4818 women), or axillary clearance versus radiotherapy as adjuncts to mastectomy (4370 women). Conclusions Some of the local therapies for breast cancer had substantially different effects on the rates of local recurrence — such as the reduced recurrence with the addition of radiotherapy to surgery — but there were no definite differences in overall survival at 10 years.

Background There have been many randomised trials of adjuvant tamoxifen among women with early breast cancer, and an updated overview of their results is presented. Methods In 1995, information was sought on each woman in any randomised trial that began before 1990 of adjuvant tamoxifen versus no tamoxifen before recurrence. Information was obtained and analysed centrally on each of 37 000 women in 55 such trials, comprising about 87% of the worldwide evidence. Compared with the previous such overview, this approximately doubles the amount of evidence from trials of about 5 years of tamoxifen and, taking all trials together, on events occurring more than 5 years after randomisation. Findings Nearly 8000 of the women had a low, or zero, level of the oestrogen-receptor protein (ER) measured in their primary tumour. Among them, the overall effects of tamoxifen appeared to be small, and subsequent analyses of recurrence and total mortality are restricted to the remaining women (18 000 with ER-positive tumours, plus nearly 12 000 more with untested tumours, of which an estimated 8000 would have been ER-positive). For trials of 1 year, 2 years, and about 5 years of adjuvant tamoxifen, the proportional recurrence reductions produced among these 30 000 women during about 10 years of follow-up were 21% (SD 3), 29% (SD 2), and 47% (SD 3), respectively, with a highly significant trend towards greater effect with longer treatment (χ21=52·0, 2p Interpretation For women with tumours that have been reliably shown to be ER-negative, adjuvant tamoxifen remains a matter for research. However, some years of adjuvant tamoxifen treatment substantially improves the 10-year survival of women with ER-positive tumours and of women whose tumours are of unknown ER status, with the proportional reductions in breast cancer recurrence and in mortality appearing to be largely unaffected by other patient characteristics or treatments.

CDK7 inhibition has emerged as a promising strategy in a range of cancer indications. CDK7 acts as a regulator of transcription through its role in phosphorylating and activating RNA Polymerase II (polII) as well as a master regulator of the cell cycle through phosphorylation of members of the CDK family. Inhibition of CDK7 in cancer cells has been shown to be effective in blocking transcriptional addiction to a defined cluster of genes (Wang et al 2015) and leads to decreased cell cycling, a decrease in the levels of proto-oncogenes such as c-Myc and induction of apoptosis. Here we describe the properties of a potent, selective and orally bioavailable CDK7 inhibitor CT7001 (formerly known as ICEC0942). Profiling across a pan-cancer cell line panel showed that CT7001 has anti-proliferative activity in a range of cancer types including acute myeloid leukaemia (AML), small cell lung (SCL) and hormone sensitive and triple negative breast cancers (TNBC).Mechanistic investigations revealed that CT7001s activity was associated with both a decrease in cell proliferation and an induction of apoptosis. Inhibition of cell proliferation was commensurate with a decrease in the levels of c-Myc, Mcl-1 and phospho-Pol II as determined by Western Blot analysis.CT7001 was then evaluated in a range of pre-clinical cancer models. CT7001 was shown to lead to cell death in a panel of patient derived models of small-cell lung cancer (SCLC) in 3D spheroid assays. In an in vivo orthotopic-patient derived xenograft model of TNBC in nu/nu mice, orally administered CT7001 monotherapy produced strong and sustained regression of the tumour that persisted during the dosing schedule, and strong suppression was still maintained upon cessation of treatment. At doses that produced regression CT7001 was well tolerated with little effect on body weight ( Citation Format: Edward K. Ainscow, Andrew Leishman, Elaine Sullivan, Bo Li, William Gallagher, Adam Peall, Kristopher Clark, Stuart Thomson, Simak Ali, Raoul C. Coombes, Ash Bahl. CT7001: An orally bioavailable CDK7 inhibitor is a potential therapy for breast, small-cell lung and haematological cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4834.

We report the safety, biodistribution, and internal radiation dosimetry of a new PET tracer, (18)F-AH111585, a peptide with a high affinity for the alpha(v)beta(3) integrin receptor involved in angiogenesis.PET scans of 8 healthy volunteers were acquired at time points up to 4 h after a bolus injection of (18)F-AH111585. (18)F activity in whole blood and plasma and excreted urine were measured up to 4 h after injection. In vivo (18)F activities in up to 12 source regions were determined from quantitative analysis of the images. The cumulated activities subsequently calculated were then used to determine the internal radiation dosimetry, including the effective dose.Injection of (18)F-AH111585 was well tolerated in all subjects, with no serious or drug-related adverse events reported. The main route of (18)F excretion was renal (37%), and the 3 highest initial uptakes were by liver (15%); combined walls of the small, upper large, and lower large intestines (11%); and kidneys (9%). The 3 highest absorbed doses were received by the urinary bladder wall (124 microGy/MBq), kidneys (102 microGy/MBq), and cardiac wall (59 microGy/MBq). The effective dose was 26 microGy/MBq.(18)F-AH111585 is a safe PET tracer with a dosimetry profile comparable to other common (18)F PET tracers.

MicroRNA (miR)-106b~25 cluster regulates bypass of doxorubicin and γ-radiation induced senescence by downregulation of the E-cadherin transcriptional activator EP300. We asked whether upregulation of miR-106~25 cluster generates cells with a truly multidrug resistant (MDR) phenotype and whether this is due to upregulation of the ATP-binding cassette (ABC) transporter P-glycoprotein. We used minimally transformed mammary epithelial breast cancer cells (MTMECs) in which the miR-106b~25 cluster was experimentally upregulated by lentiviral transfection or in which hairpins targeting either EP300 or E-cadherin mRNAs have been expressed with lentiviruses. We find that overexpression of miR-106b~25 cluster led to the generation of MDR MTMECs (resistant to etoposide, colchicine and paclitaxel). Paclitaxel resistance was also studied after experimental downregulation of EP300 or E-cadherin. However none of these cells overexpressed P-glycoprotein or where able to efflux a fluorescent derivative of paclitaxel, making this phenotype drug-transporter independent. Paclitaxel treatment in MTMECs led to an increase in early apoptotic cells (Annexin V-positive), activation of caspase-9 and increase in the proportion of cells at the G2/M phase of the cell cycle. However, MTMEC overexpressing miR-106b~25 cluster, or with EP300 or E-cadherin downregulated, showed less activation of apoptosis, caspase-9 and caspase-3/-7 activities. Thus, miR-106b~25 cluster controls transporter-independent MDR by apoptosis evasion via downregulation of EP300.

Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch(R) system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Estrogens have long been recognized as being important for stimulating the growth of a large proportion of breast cancers. Now it is recognized that estrogen action is mediated by two receptors, and the presence of estrogen receptor alpha (ER alpha) correlates with better prognosis and the likelihood of response to hormonal therapy. Over half of all breast cancers overexpress ER alpha and around 70% of these respond to anti-estrogen (for example tamoxifen) therapy. In addition, the presence of elevated levels of ER alpha in benign breast epithelium appears to indicate an increased risk of breast cancer, suggesting a role for ER alpha in breast cancer initiation, as well as progression. However, a proportion of ER alpha-positive tumors does not respond to endocrine therapy and the majority of those that do respond eventually become resistant. Most resistant tumors remain ER alpha-positive and frequently respond to alternative endocrine treatment, indicative of a continued role for ER alpha in breast cancer cell proliferation. The problem of resistance has resulted in the search for and the development of diverse hormonal therapies designed to inhibit ER alpha action, while research on the mechanisms which underlie resistance has shed light on the cellular mechanisms, other than ligand binding, which control ER alpha function.

The cloning of a novel estrogen receptor beta (denoted ERbeta) has recently been described (Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5925-5930 and Mosselman, S., Polman, J. , and Dijkema, R. (1996) FEBS Lett. 392, 49-53). ERbeta is highly homologous to the "classical" estrogen receptor alpha (here referred to as ERalpha), has been shown to bind estrogens with an affinity similar to that of ERalpha, and activates expression of reporter genes containing estrogen response elements in an estrogen-dependent manner. Here we describe functional studies comparing the DNA binding abilities of human ERalpha and beta in gel shift assays. We show that DNA binding by ERalpha and beta are similarly affected by elevated temperature in the absence of ligand or in the presence of 17beta-estradiol and the partial estrogen agonist 4-hydroxy-tamoxifen. In the absence of ligand, DNA binding by ERalpha and beta is rapidly lost at 37 degrees C, while in the presence of 17beta-estradiol and 4-hydroxy-tamoxifen, the loss in DNA binding at elevated temperature is much more gradual. We show that the loss in DNA binding is not due to degradation of the receptor proteins. However, while the complete antagonist ICI 182, 780 does not "protect" human ERalpha (hERalpha) from loss of DNA binding at elevated temperature in vitro, it does appear to protect human ERbeta (hERbeta), suggestive of differences in the way ICI 182, 780 acts on hERalpha and beta. We further report that ERalpha and beta can dimerize with each other, the DNA binding domain of hERalpha being sufficient for dimerization with hERbeta. Cell and promoter-specific transcription activation by ERalpha has been shown to be dependent on the differential action of the N- and C-terminal transcription activation functions AF-1 and AF-2, respectively. The existence of a second estrogen receptor gene and the dimerization of ERalpha and beta add greater levels of complexity to transcription activation in response to estrogens.

Resistance to chemotherapeutic treatment, which is indirectly responsible for many cancer deaths, is normally associated with an aggressive phenotype including increased cell motility and acquisition of invasive properties. Here we describe how breast cancer cells overcome doxorubicin-induced senescence and become drug resistant by overexpression of the microRNA (miR)-106b~25 cluster. Although all three miRs in the cluster contribute to the generation of doxorubicin resistance, miR-25 is the major contributor to this phenotype. All three miRs in this cluster target EP300, a transcriptional activator of E-cadherin, resulting in cells acquiring a phenotype characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT), including an increase in both cell motility and invasion, as well as the ability to proliferate after treatment with doxorubicin. These findings provide a novel drug resistance/EMT regulatory pathway controlled by the miR-106b~25 cluster by targeting a transcriptional activator of E-cadherin.

Regulation of gene expression by sequence-specific DNA binding proteins involves the co-ordinated action of a repertoire of transcriptional coregulator complexes, which together act to modify chromatin at gene promoters, thereby facilitating gene expression. The mechanisms by which such coregulators are recruited to the promoters of estrogen-responsive genes by estrogen receptor-\upalpha have been well studied in breast cancer cells. These studies have highlighted coactivator and corepressor proteins that appear to be critical for the agonist and antagonist actions of estrogen and anti-estrogens, and indicate that altered levels and/or activities of these proteins is an important feature of response and resistance to endocrine treatments in breast cancer.

Background: [18F]ICMT-11 is an isatin analogue which has been developed by our group as a novel PET radiotracer for studies of apoptosis in vivo. Preclinical studies have demonstrated subnanomolar affinity to caspase 3, and validated the potential for imaging apoptosis in xenograft models. A first-in-man study showed that the agent was well tolerated with acceptable dosimetry. This is the first study of this agent to measure the effect of chemotherapy on radiotracer uptake in patients. As apoptosis is a dynamic process, one of the main objectives of the study was to determine the optimal time-point for imaging post-chemotherapy and compare the results with immunohistochemistry assessments at the same time-points. The study was approved by a regional ethics committee and ARSAC. Methods: 7 patients with breast tumour lesions measuring 15mm or more, due to undergo neoadjuvant chemotherapy with FEC (5FU, Epirubicin, Cyclophosphamide) had dynamic PET scans for 66mins 30seconds following intravenous injection of [18F]ICMT-11 with a mean activity of 340.82 Mbq±20.76 and Specific Activity range of 447.014-5128.34 Gbq/µmol prior to chemotherapy and 24h-2 weeks post-chemotherapy. A breast biopsy was also obtained within a few hours of the 2nd PET scan to correlate apoptosis in the breast tissue utilising TUNEL and Caspase 3 staining by immunohistochemistry. Volumes of interest were drawn manually and analyzed using Analyze software®. Results: The scans were well tolerated in all patients. Uptake of [18F]ICMT-11 was demonstrated in all tumor lesions. The tumours studied included ER positive and PR positive, HER2 positive and triple negative patients. The first cohort patients were imaged pre-chemotherapy and 24-48h post chemotherapy. Tumour to Breast ratio (TBR) showed an increase from 1.42±0.21(pre) to 1.71±0.33 (post). Tumour to muscle ratio (TMR) was not increased, 1.52±0.30(pre) and 1.22±0.09 (post). In addition, an increase the SUV was noted in the lymph nodes of patients, at both 24 and 48h. (SUVav 0.39±0.02 (pre), 0.45±0.03 (post), and SUVmax 0.87± 0.02(pre) ,1.22±0.12 (post). The lymph nodes were however not sampled for immunohistochemistry. A further cohort of patients had the follow-up scan 2 weeks post chemotherapy, TBR and TMR were both increased in this cohort 1.50±0.22 (pre), 2.52±0.48 (post),and 1.82±0.10 (pre), 2.08±0.0.04 (post), respectively. Caspase and TUNEL labelling with immunohistochemistry also showed increased in apoptosis in the breast biopsies at 2 weeks compared to baseline in keeping with the PET data. Conclusion: These preliminary data suggest that [18F]ICMT-11 is a promising marker for chemotherapy induced apoptosis in vivo, and correlates with findings in tumor biospsies using TUNEL and immunostaining for caspase 3. Further work is underway to study a larger cohort of patients, and identify the optimal PET pharmacokinetic parameter to describe [18F]ICMT-11 uptake and retention. Citation Format: Shairoz Merchant, Eric O Aboagye, Adrian Lim, Kasia Kozlowski, Naina Patel, Jennifer Steel, Susan Cleator, Sami Shousha, Vidhya Varghese, Raoul C Coombes, Laura Kenny. Evaluation of apoptosis in breast cancer using the novel PET probe [18F]ICMT-11 in patients treated with neoadjuvant FEC chemotherapy: Initial assessment of optimum imaging time and relation to caspase-3 immunostaining [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-01-02.

The objective of this study was to evaluate the clinical response of locally advanced breast cancer (LABC) to neoadjuvant (NA) chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide (FEC) and to study the role of docetaxel in patients who fail to respond to first-line chemotherapy. Patients were enrolled who had primary tumours without distant metastasis that were too extensive for conservative surgery. All underwent NA chemotherapy for breast cancer and thereafter surgery and/or radical radiotherapy. NA chemotherapy with FEC was administered to 88 patients between February 1998 and June 2005. A median of 6 cycles of FEC (range 1-8) was given, followed in 21 cases by a median of 4 cycles (range 2-6) of docetaxel. Where clinically established, with FEC the clinical complete response (cCR) was 22/81 (27%), clinical partial response (cPR) 41/81 (51%), clinical stable disease (cSD) 18/81 (22%). In patients where the response to FEC was regarded as insufficient, docetaxel was given. Response rates were cCR 3/21 (14%); cPR 10/21 (48%), cSD 8/21 (38%). There were 11 cases of pathological complete response (pCR), 9 in the FEC-only group and 2 in the docetaxel group. Following chemotherapy 49 (56%) patients underwent mastectomy, 32 (36%) breast conserving surgery and 5 (6%) radical radiotherapy, giving a breast conservation rate of 42%. Two patients died before receiving surgery or radical radiotherapy. The results show that neoadjuvant FEC is a reasonable NA therapy in breast cancer and that docetaxel is effective in FEC refractory cases. Only 8 of 81 (10%) assessable patients did not respond to any chemotherapy, giving an overall clinical response rate of 90%, which is comparable to studies in which taxanes were given irrespective of response to preceding therapy with antracycline including regimes.

Trichostatin A is a potent and specific histone deacetylase inhibitor with promising antitumor activity in preclinical models. Plasma pharmacokinetics of trichostatin A were studied following single-dose intraperitoneal administration of 80 mg/kg (high dose) or 0.5 mg/kg (low dose) to female BALB/c mice. Plasma trichostatin A concentrations were quantified by high performance liquid chromatography (HPLC)-UV assay (high dose) or by HPLC-multiple reaction monitoring assay (low dose). Trichostatin A was rapidly absorbed from the peritoneum and detectable in plasma within 2 min. Cmax of 40 microg/ml and 8 ng/ml occurred within 5 min, followed by rapid exponential decay in plasma trichostatin A concentration with t1/2 of 6.3 min and 9.6 min (high and low doses, respectively). Phase I metabolites at the high dose were identified by simultaneous UV and positive ion electrospray mass spectrometry. Trichostatin A underwent extensive metabolism: primary metabolic pathways were N-demethylation, reduction of the hydroxamic acid to the corresponding trichostatin A amide, and oxidative deamination to trichostatic acid. N-Monomethyl trichostatin A amide was the major plasma metabolite. No didemethylated compounds were identified. Trichostatic acid underwent further biotransformation: reduction and beta-oxidation of the carboxylic acid, with or without N-demethylation, resulted in formation of dihydro trichostatic acid and dinor dihydro trichostatic acids. HPLC fractions corresponding to trichostatin A and N-demethylated trichostatin A exhibited histone deacetylase-inhibitory activity; no other fractions were biologically active. We conclude that trichostatin A is rapidly and extensively metabolized in vivo following intraperitoneal administration to mice, and N-demethylation does not compromise histone deacetylase-inhibitory activity.

Background: Nicastrin (NCT) is the gate keeper of the gamma secretase (GS) enzyme complex. We have previously shown that high levels of NCT are observed in breast cancer patients. Moreover, we recently highlighted the importance of NCT expression for the expansion of breast cancer stem cells and their invasive features. The involvement of NCT in development of chemoresistance has been suggested by the evidence that 1) NCT is increased in response to oxaliplatin, irinotecan and 5-FU in colon cancer and 2) NCT regulates cell death within the p53 and AKT signalling pathway in human embryonic kidney (HEK293) cells. Here, we have explored the potential role of NCT in the chemoresistance of breast cancer cells. Methods: Stable NCT overexpressing MCF10A cells and control cells were produced by retroviral infections. siRNA strategy was used to silence NCT expression in a panel of p53wt and p53mut breast cancer cell lines. The sulphorhodamine B and Annexin V/PI staining were used to measure proliferation and apoptosis rate in breast cancer cells unstressed or treated with chemotherapeutic agents alone or in combination with DAPT, Wortmannin or Nutlin. p53 levels and activity were assessed by Western blotting, RT-qPCR and gene reporter assays. Results: Stable overexpression of NCT in MCF10A cells induced resistance to doxorubicin and cisplatin treatments as demonstrated by a decreased early and late apoptotis. Moreover, NCT lowers p53 expression, its phosphorylation at the phosphoSer15 site and its transcriptional activity in both unstressed cells and doxorubicin-treated cells. Using DAPT and Wortmannin as pharmacological inhibitors of Notch and AKT signalling pathways and Nutlin to inhibit Mdm2-mediated p53 degradation, we confirmed that NCT mediated chemoresistance in a p53 dependent manner partially through the PI3K/AKT pathways, while it is role is completely independent from GS activity. NCT knockdown was able to sensitize several breast cancer cell lines (MCF7, T47D, ZR75) with a wild type p53 status to doxorubicin. Moreover, NCT overexpression reduced the sensitivity to cisplatin in p53wt HTC116 colon cancer cells while it had no effect on p53mut cells. In order to translate these results to clinical settings, we have developed anti-NCT monoclonal antibodies which have been showed to inhibit proliferation and invasion of breast cancer cells. These antibodies used in combination with chemotherapy mirrored the positive effect of NCT siRNA in terms of response to doxorubicin in p53wt breast cancer cell lines. Conclusions: Our study indicates that in breast cancer cells NCT controls chemotherapy-induced cell death in a p53-dependent manner through the regulation of AKT pathway. This function is independent from the GS. We aim to investigate the association of NCT expression and p53 mutation status with clinical outcome in breast cancers in order to support potential combination of anti-NCT monoclonal antibodies with chemotherapy. Citation Format: Ylenia Lombardo, Aleksandra Filipovic, Monica Faronato, Raoul Charles Coombes. Role of nicastrin in the mediating chemoresistance of breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 829. doi:10.1158/1538-7445.AM2013-829

Background Brain metastases are common in human epidermal growth factor receptor (Her)-2-positive breast cancer. Drug access to brain metastases and normal brain is key to management of cranial disease. In this study, positron emission tomography (PET) scanning after administration of radiolabelled lapatinib was used to obtain direct evidence of cranial drug access. Methods Patients with Her-2+ metastatic breast cancer either with at least one 1-cm diameter brain metastasis or without brain metastases underwent dynamic carbon-11 radiolabelled lapatinib ([11C]lapatinib)-PET. Less than 20 μg of [11C]lapatinib was administered before and after 8 days of oral lapatinib (1,500 mg once daily). Radial arterial blood sampling was performed throughout the 90-min scan. The contribution of blood volume activity to the tissue signal was excluded to calculate lapatinib uptake in normal brain and metastases. Partitioning of radioactivity between plasma and tissue (V T) was calculated and the tissue concentration of lapatinib derived. Plasma lapatinib levels were measured and adverse events noted. Results Six patients (three with brain metastases) were recruited. About 80% plasma radioactivity corresponded to intact [11C]lapatinib after 60 min. PET signal in the brain corresponded to circulating radioactivity levels, with no [11C]lapatinib uptake observed in normal brain tissue. In contrast, radioactivity uptake in cranial metastases was significantly higher (p = 0.002) than that could be accounted by circulating radioactivity levels, consistent with [11C]lapatinib uptake in brain metastases. There was no difference in lapatinib uptake between the baseline and day 8 scans, suggesting no effect of increased drug access by inhibition of the drug efflux proteins by therapeutic doses of lapatinib. Conclusions Increased lapatinib uptake was observed in brain metastases but not in normal brain. Trial registration ClinicalTrials.gov: NCT01290354