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Background: 80% of all breast cancers (BCs) are ER-positive (ER+). Not all respond to adjuvant endocrine therapy (aET) and a significant number develop endocrine resistance and recur. The basis for primary and acquired endocrine resistance is poorly understood. A multiomics analysis of primary ER+ BCs matched with recurrences on or after completion of aET has been performed. Patients: A unique cohort of 520 women with matched primary and recurrent ER+, HER2-negative (HER2-) BC is being analysed. In the first subset of 75, all had surgery to clear margins, followed by aET. The endocrine therapy given was tamoxifen (66%), aromatase inhibitors (AI) (28%: 17% letrozole, 6% anastrozole, 2% exemestane, and 3% a succession of 2 different AIs), or a combination of tamoxifen and an AI (6%). aET duration was 5 years, unless the patient stopped treatment or developed a recurrence sooner. 16/75 patients (21%) had positive lymph nodes. All patients developed recurrences: local in 59/75, concurrent local and nodal in 13/75 and lymph node-only in 3/75. Median time to recurrence was 4.1 years (range: 0.7-29 years). 62% of patients were on aET at the time of recurrence. All patients have long-term follow-up. Methods: DNA and RNA were extracted from matched primary and recurrence BC tissue samples. Targeted DNA-exome and whole-genome expression analyses were performed. A custom targeted DNA panel was used to study genes implicated in endocrine therapy resistance (ETR): this included 73 different targets, selected based on our previous full-exome sequencing of sequential ET recurrences and those implicated in the literature and in curated somatic and cancer mutation databases. Somatic mutations and copy number alterations (CNA) were determined. Differential gene expression analysis was performed using two-class unpaired Significance Analysis of Microarrays (SAM). Validation of pathways implicated in ETR using NanoString GeoMx protein analysis is ongoing. Results: Targeted DNA-exome profiling identified 1 or 2 potential driver mutations in all but a few primary samples. Multiple aberrations and a highly diverse mutational landscape were observed in all the recurrences. Matched breast and lymph node samples from synchronous recurrences had very similar somatic profiles. Changes significantly enriched in recurrent samples included somatic aberrations in well-established drivers such as MAP3K1, PIK3CA, TP53 and CDH1, as well as ESR1. Aberrations were also common in PTEN and in ER-associated factors FOXA1 and GATA3. Transcriptomic analysis revealed a number of pathways implicated in resistance, including ER, HER2, GATA3, AKT, RAS and p63 signalling. A panel-based, targeted DNA sequencing approach for mutational profiling allowed capture of relevant mutational profiles linked to ETR in a cost-effective manner compared with traditional whole-exome sequencing. Ongoing analysis has linked mutational profiles to specific endocrine agents and has allowed us to demonstrate significant differences between recurrences on aET compared with those after completion of aET. Multiomics profiling of the remaining samples in the cohort is underway. Discussion: This multiomics study provides the largest cohort to-date of matched early and recurrent ER+/HER2- BCs. It has shed new light on how different adjuvant endocrine agents can affect primary drivers and lead to complex somatic and transcriptomic changes in recurrent disease. This work confirms that the mechanisms of endocrine resistance are diverse and has already identified mechanisms underlying ETR and clinically meaningful biomarkers of ETR, including potentially actionable mutations and targets. Citation Format: Carlos Martinez-Perez, Charlene Kay, James Meehan, J Michael Dixon, Arran K Turnbull. PD10-09 Multiomics analysis of matched ER+ primary and recurrent breast cancers on or after adjuvant endocrine therapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-09.

Supplementary Data from Integrated DNA and RNA Sequencing Reveals Drivers of Endocrine Resistance in Estrogen Receptor–Positive Breast Cancer

Purpose:Endocrine therapy resistance (ETR) remains the greatest challenge in treating patients with hormone receptor–positive breast cancer. We set out to identify molecular mechanisms underlying ETR through in-depth genomic analysis of breast tumors.Experimental Design:We collected pre-treatment and sequential on-treatment tumor samples from 35 patients with estrogen receptor–positive breast cancer treated with neoadjuvant then adjuvant endocrine therapy; 3 had intrinsic resistance, 19 acquired resistance, and 13 remained sensitive. Response was determined by changes in tumor volume neoadjuvantly and by monitoring for adjuvant recurrence. Twelve patients received two or more lines of endocrine therapy, with subsequent treatment lines being initiated at the time of development of resistance to the previous endocrine therapy. DNA whole-exome sequencing and RNA sequencing were performed on all samples, totalling 169 unique specimens. DNA mutations, copy-number alterations, and gene expression data were analyzed through unsupervised and supervised analyses to identify molecular features related to ETR.Results:Mutations enriched in ETR included ESR1 and GATA3. The known ESR1 D538G variant conferring ETR was identified, as was a rarer E380Q variant that confers endocrine hypersensitivity. Resistant tumors which acquired resistance had distinct gene expression profiles compared with paired sensitive tumors, showing elevated pathways including ER, HER2, GATA3, AKT, RAS, and p63 signaling. Integrated analysis in individual patients highlighted the diversity of ETR mechanisms.Conclusions:The mechanisms underlying ETR are multiple and characterized by diverse changes in both somatic genetic and transcriptomic profiles; to overcome resistance will require an individualized approach utilizing genomic and genetic biomarkers and drugs tailored to each patient.

Supplementary Figure from Integrated DNA and RNA Sequencing Reveals Drivers of Endocrine Resistance in Estrogen Receptor–Positive Breast Cancer

While the term biomarker is thought to have first been used in the 1970s, the concept itself is considered to be much older [...]

IL6-like cytokines are a family of regulators with a complex, pleiotropic role in both the healthy organism, where they regulate immunity and homeostasis, and in different diseases, including cancer. Here we summarise how these cytokines exert their effect through the shared signal transducer IL6ST (gp130) and we review the extensive evidence on the role that different members of this family play in breast cancer. Additionally, we discuss how the different cytokines, their related receptors and downstream effectors, as well as specific polymorphisms in these molecules, can serve as predictive or prognostic biomarkers with the potential for clinical application in breast cancer. Lastly, we also discuss how our increasing understanding of this complex signalling axis presents promising opportunities for the development or repurposing of therapeutic strategies against cancer and, specifically, breast neoplasms.

Background: Radiotherapy (RT) plays an important role in the multimodal treatment of breast cancer (BC). Despite improvements in the accuracy of delivering radiation to specific biological target volumes, the clinical response of BC to RT is still affected by intrinsic/acquired radioresistance. These resistant cancer cells can contribute to the development of recurrent disease and poor patient outcomes. Clinical signs of RT response are often not apparent for several weeks post-treatment; patients who fail to respond will therefore initially go undetected. There are currently no clinically validated biomarkers that can predict which patients will respond to RT or assess response during treatment. Our study aims to address this major clinical need through the identification and validation of biomarkers of radiation responsiveness. Methods: The effects of different radiation doses (2 - 10Gy) at a range of time points (1 - 24h) were investigated by analysing the protein secretion profiles from 3 BC cell lines: MCF-7 (ER+), ZR-751 (ER+) and MDA-MB-231 (ER-). Conditioned media was collected from each dose/time point and proteins isolated for mass spec analysis. For comparison, radioresistant models were derived from each of the 3 cell lines and were characterized by proliferation and colony formation assays, invasion and migration assays, whole-genome transcriptomic sequencing (WGTS) analysis and western blotting. To assess intrinsic response to RT a panel of 16 BC cell lines were evaluated by colony formation assays following a 2Gy dose of radiation. WGTS of a patient cohort of 230 (138 ER+ve, 92 ER-ve) post-menopausal women with BC, treated with breast conserving surgery and adjuvant RT but no systemic adjuvant therapy, with a median follow-up of 14 years, is currently underway. Results: 9 biomarkers emerged whose secretion was significantly increased with radiation. These were evaluated by western blotting of conditioned media 24h after a 2Gy dose of RT in matched radio-sensitive and resistant cell lines; this confirmed significantly higher levels of radiation induced secretion from sensitive cells compared to resistant. Radioresistant cell lines were characterised by epithelial-to-mesenchymal-transition, enhanced invasion/migration, loss of ER and PgR and increased EGFR and PI3K signaling activity. Initial mechanistic investigations suggest that biomarker release in response to radiation occurs via microvesicles. A blood-based assay to test the level of these secreted biomarkers is currently under development. Pre-treatment levels of the 9 biomarkers were also found to be associated with prediction of intrinsic response to RT at both gene and protein level. A gene expression signature comprising the 9 candidates is strongly associated with the intrinsic response to RT across the 16 BC cell lines studied, with higher expression found in those more sensitive to RT compared with those less sensitive or resistant. Validation of the predictive power of these biomarkers in terms of recurrence-free and overall BC specific survival is currently being assessed at gene and protein level in the patient cohort. Conclusions: We have identified 9 biomarkers which are released from BC cells sensitive to radiation 24h after a 2Gy dose (in line with current clinical standards) but not from radio-resistant derivatives.A blood based assay is currently under development which has the potential to monitor response to RT in the neoadjuvant and palliative settings.Intracellular levels of the 9 biomarkers are strongly associated with intrinsic response to RT and may hold predictive potential.These biomarkers may have the potential to improve patient care by identifying patients less likely to benefit from RT, paving the way for personalization of treatment, including altered dosing schedules and the future use of emerging radio-sensitizers. Citation Format: James Meehan, Mark Gray, Carlos Martinez-Perez, Charlene Kay, J Michael Dixon, Jimi Wills, Carol Ward, Alex von Kriegsheim, Niall Quinn, Olga Oikonomidou, David Cameron, Simon P Langdon, David Argyle, Ian H Kunkler, Arran K Turnbull. Development and validation of novel biomarkers of response to radiotherapy in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-18.

Background: Ductal carcinoma in situ (DCIS) lesions are non-obligate precursors to invasive breast cancer (IBC). With the ultimate goal of preventing the development of invasive disease, DCIS is typically treated by breast-conserving surgery (BCS). Adjuvant radiotherapy (RT) is given for high-grade disease to reduce the risk of in-breast tumour recurrence (IBTR). The use of endocrine therapy (ET) for DCIS varies, as studies show a modest benefit but no survival improvement; in the UK, guidelines recommend ET for DCIS in some scenarios but only instead of, rather than in addition to, RT. This project sought to characterise the biology of ER+ DCIS and identify a group of women who are likely to gain the most benefit from the addition of adjuvant ET. Patients: Cohort A - 77 women with ER+ (Allred 7/8) high-grade DCIS treated with BCS plus RT, 20 of whom developed IBTR.Cohort B - 70 women with ER+ (Allred 7/8) low/intermediate-grade DCIS treated with BCS alone, 12 of whom developed IBTR.Cohort C - 68 women with ER+ (Allred 7/8) DCIS treated with BCS plus ET. All patients were treated locally between 2000 and 2016 and the median follow-up is 6 years. Methods: We performed whole-genome transcriptomic QuantSeq sequencing of samples from cohort A. Sequencing of cohort B and C is currently underway. IL6ST levels were validated using immunohistochemistry and RNAScope. Results: In cohort A, only a subset (34/77) of tumours had gene expression profiles consistent with active ER signalling. Levels of IL6ST, a biomarker for ET response, could differentiate these two subgroups and this was validated at protein level using immunohistochemistry. The low ER signalling subgroup were associated with higher levels of EGFR, HER2 and MAPK signalling. 20/77 high-grade DCIS cases recurred within 10 years. 50% of these recurred as IBC (rather than DCIS) and these were associated with higher levels of IL6ST, had active ER signalling and higher levels of proliferation-associated and estrogen receptor target genes, known to be decreased by ET, in the primary DCIS lesion. Discussion: Our findings suggest that some high-grade ER+ DCIS patients have active ER signalling while in others ER signalling remains low despite highly expressing the ER protein. IL6ST, a biomarker of endocrine therapy response can be used to differentiate these two groups of ER+ DCIS. DCIS lesions which recurred as IBC had active ER signalling and also higher levels of proliferation genes known to be decreased by ET compared with DCIS which recurred as further DCIS. These findings suggest that IL6ST may have a role in identifying a subset of ER+ DCIS which are at a higher risk of developing advanced disease and are also more likely to benefit from the addition of adjuvant ET, with a better risk-to-benefit ratio than observed in previous studies that considered a less targeted use of this treatment strategy, thus potentially reducing the risk of IBC recurrence. These findings will be validated in a cohort of low/intermediate-grade DCIS who received no adjuvant RT (cohort B) and a cohort of patients who received adjuvant ET as part of their treatment (cohort C). Citation Format: Carlos Martinez-Perez, Charlene Kay, Rebecca Swan, Gregory E Ekatah, Laura M Arthur, James Meehan, Mark Gray, Andrew H Sims, Olga Oikonomidou, Arran K Turnbull, J Michael Dixon. IL6ST, a biomarker of endocrine therapy response, has potential in identifying a subgroup of women with ER+ DCIS who are more likely to benefit from adjuvant endocrine therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-16-04.

Background: The ER+/HER2+ subtype accounts for up to 10% of all breast cancers (BCs) and most are treated with surgery followed by adjuvant chemotherapy with Herceptin +/- radiotherapy then adjuvant endocrine therapy (ET) to reduce the recurrence risk. Despite this it is clear that not all ER+/HER2+ patients gain benefit from the addition of chemotherapy and Herceptin. In particular, given the significant side effects associated with the chemotherapy, the risks may out way the benefit in some older patients and in those with co-morbidities. Currently there are no clinically validated tools to identify women with ER+/HER2+ BC whose risk of recurrence remains unchanged with the addition of chemotherapy plus Herceptin and who can be effectively managed with adjuvant ET alone. Using levels of IL6ST, a biomarker for ET response, ER+/HER2+ patients who gain benefit from ET can be differentiated from those for whom ET alone is not sufficient to reduce the risk of recurrence. The latter group were characterised by inactive ER signalling and active MAPK and PI3K signalling. The aim is to show that ER+/HER2+ patients predicted to respond less well to ET alone using IL6ST levels, gain additional benefit from chemotherapy plus Herceptin. Patients: • Cohort A - 32 post-menopausal women (PMW) with large ER+/HER2+ BC treated with neoadjuvant (3-6 months) then adjuvant letrozole. 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. Median follow-up was 7.5 years. • Cohort B - 362 women with ER+/HER2+ BC treated with surgery +/- radiotherapy followed by adjuvant endocrine therapy between 2005 and 20010. 219 also received chemotherapy plus Herceptin. Median follow-up is 9.5 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 acquired resistance after a period of response. Neoadjuvant clinical response was predicted with 92% accuracy using levels of IL6ST. Gene expression analysis in 17 patients showed increased MAPK and PI3K pathway activity in the 9 NR compared with the 8 R tumours. 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. Samples from cohort B are currently being profiled using IL6ST in tandem with custom gene expression assays for ER, MAPK and PI3K pathway activity. Conclusions: • IL6ST levels can differentiate ER+/HER2+ BCs who respond well to ET alone and those with a poor clinical response who have a higher risk of recurrence. • NR to ET have increased expression of PI3K and MAPK pathways, consistent with active HER2 signalling. • Analysis of cohort B is underway and will elucidate the benefit in terms of recurrence-free and overall breast cancer specific survival from the addition of chemotherapy plus Herceptin to the group predicted to respond less well to ET using IL6ST. • There is potential role for IL6ST in selecting ER+/HER2+ patients that require and benefit from HER2-targeted therapies. Citation Format: Arran K Turnbull, Victoria Webber, Daniel McStay, Laura M Arthur, Carlos Martinez-Perez, James Meehan, Mark Gray, Charlene Kay, Lorna Renshaw, Jane Keys, Robert Clarke, Andrew H Sims, Olga Oikonomidou, J Michael Dixon. Can some ER+/HER2+ patients be safely spared from treatment with chemotherapy plus herceptin? [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-07.

Recent advances in the fields of electronics and microfabrication techniques have led to the development of implantable medical devices for use within the field of precision medicine. Monitoring visceral surface tissue O2 tension ([Formula: see text]) by means of an implantable sensor is potentially useful in many clinical situations, including the perioperative management of patients undergoing intestinal resection and anastomosis. This concept could provide a means by which treatment could be tailored to individual patients. This study describes the in vivo validation of a novel, miniaturized electrochemical O2 sensor to provide real-time data on intestinal [Formula: see text]. A single O2 sensor was placed onto the serosal surface of the small intestine of anesthetized rats that were exposed to ischemic (superior mesenteric artery occlusion) and hypoxemic (alterations in inspired fractional O2 concentrations) insults. Control experiments demonstrated that the sensors can function and remain stable in an in vivo environment. Intestinal [Formula: see text] decreased following superior mesenteric artery occlusion and with reductions in inspired O2 concentrations. These results were reversible after reinstating blood flow or by increasing inspired O2 concentrations. We have successfully developed an anesthetized rat intestinal ischemic and hypoxic model for validation of a miniaturized O2 sensor to provide real-time measurement of intestinal [Formula: see text]. Our results support further validation of the sensors in physiological conditions using a large animal model to provide evidence of their use in clinical applications where monitoring visceral surface tissue O2 tension is important. NEW & NOTEWORTHY This is the first report of real-time continuous measurements of intestinal oxygen tension made using a microfabricated O2 sensor. Using a developed rodent model, we have validated this sensor's ability to accurately measure dynamic and reversible changes in intestinal oxygenation that occur through ischemic and hypoxemic insults. Continuous monitoring of local intestinal oxygenation could have value in the postoperative monitoring of patients having undergone intestinal surgery.

Worldwide, prostate cancer (PC) is the second-most-frequently diagnosed male cancer and the fifth-most-common cause of all cancer-related deaths. Suspicion of PC in a patient is largely based upon clinical signs and the use of prostate-specific antigen (PSA) levels. Although PSA levels have been criticised for a lack of specificity, leading to PC over-diagnosis, it is still the most commonly used biomarker in PC management. Unfortunately, PC is extremely heterogeneous, and it can be difficult to stratify patients whose tumours are unlikely to progress from those that are aggressive and require treatment intensification. Although PC-specific biomarker research has previously focused on disease diagnosis, there is an unmet clinical need for novel prognostic, predictive and treatment response biomarkers that can be used to provide a precision medicine approach to PC management. In particular, the identification of biomarkers at the time of screening/diagnosis that can provide an indication of disease aggressiveness is perhaps the greatest current unmet clinical need in PC management. Largely through advances in genomic and proteomic techniques, exciting pre-clinical and clinical research is continuing to identify potential tissue, blood and urine-based PC-specific biomarkers that may in the future supplement or replace current standard practices. In this review, we describe how PC-specific biomarker research is progressing, including the evolution of PSA-based tests and those novel assays that have gained clinical approval. We also describe alternative diagnostic biomarkers to PSA, in addition to biomarkers that can predict PC aggressiveness and biomarkers that can predict response to certain therapies. We believe that novel biomarker research has the potential to make significant improvements to the clinical management of this disease in the near future.

Novel biomarkers are needed to continue to improve breast cancer clinical management and outcome. IL6-like cytokines, whose pleiotropic functions include roles in many hallmarks of malignancy, rely on the signal transducer IL6ST (gp130) for all their signalling. To date, 10 separate independent studies based on the analysis of clinical breast cancer samples have identified IL6ST as a predictor. Consistent findings suggest that IL6ST is a positive prognostic factor and is associated with ER status. Interestingly, these studies include 4 multigene signatures (EndoPredict, EER4, IRSN-23 and 42GC) that incorporate IL6ST to predict risk of recurrence or outcome from endocrine or chemotherapy. Here we review the existing evidence on the promising predictive and prognostic value of IL6ST. We also discuss how this potential could be further translated into clinical practice beyond the EndoPredict tool, which is already available in the clinic. The most promising route to further exploit IL6ST’s promising predicting power will likely be through additional hybrid multifactor signatures that allow for more robust stratification of ER+ breast tumours into discrete groups with distinct outcomes, thus enabling greater refinement of the treatment-selection process.

Treatment for HR+/HER2+ patients has been debated, as some tumors within this luminal HER2+ subtype behave like luminal A cancers, whereas others behave like non-luminal HER2+ breast cancers. Recent research and clinical trials have revealed that a combination of hormone and targeted anti-HER2 approaches without chemotherapy provides long-term disease control for at least some HR+/HER2+ patients. Novel anti-HER2 therapies, including neratinib and trastuzumab emtansine, and new agents that are effective in HR+ cancers, including the next generation of oral selective estrogen receptor downregulators/degraders and CDK4/6 inhibitors such as palbociclib, are now being evaluated in combination. This review discusses current trials and results from previous studies that will provide the basis for current recommendations on how to treat newly diagnosed patients with HR+/HER2+ disease.

Background:In breast cancer (BC), radiotherapy (RT) is used adjuvantly to prevent recurrence and also in the palliative setting. Clinical signs of RT response are often not apparent for several weeks post-treatment and we currently lack tools to predict or monitor tumor response to RT early during treatment. The aim was to identify tumor-secreted biomarkers whose release reflects response to RT, which could be monitored during treatment in the blood or intratumorally by an implantable biosensor, currently under development within the Implantable Microsystems for Personalised Anti-Cancer Therapy (IMPACT) program. Methods: A series of experiments assessed the effect of different radiation doses (2-10Gy) on 3 human BC cell lines – MDA-MB-231 (ER-), MCF-7 (ER+) and HBL-100 (ER-) –, 1 canine breast cancer and 2 sheep lung cancer lines. Culture media was collected from each dose experiment at a range of post-radiation time-points (1-24 hours). Proteins were isolated from collected media for secretome mass spectrometry (MS) analysis. A subset of treatment/time conditions were repeated in the same BC cell lines and radioresistant (RR) derivatives from which RNA was extracted and analysed using Lexogen QuantSeq for whole-genome transcriptomics.In-lab candidate biomarker validation was carried out using immuhistochemistry (IHC), immunofluorescence (IF) and western blotting (WB) using validated antibodies. Levels of candidate biomarkers were also assessed in normal and untreated BC tissues using IHC. ELISA-based methods are currently under investigation for detection of the lead candidate biomarkers in the blood of large animal cancer models treated with RT. Results: Biomarker discovery using the MS data revealed 4 promising candidates: EIF3G, SEC24C, YBX3 and TK1. These are released from BC and animal cancer cells sensitive to radiation in a dose-dependent manner 24 hours after treatment. Analysis of the transcriptomic data showed an 8-fold higher expression of the genes encoding the 4 candidates in the radio-sensitive parental cell lines compared to the RR cell lines. IF and WB confirmed lower intracellular expression of the 4 proteins in RR cells compared to the parental lines. WB of collected culture media confirmed release of each of the 4 candidates 24 hours after a 2Gy dose of radiation in only the parental lines. GAPDH was not found in these media samples, demonstrating that protein release was not due to cell lysis. Conclusions: · We have identified 4 promising biomarkers which are released from cancer cells sensitive to RT and not released from RR derivatives. · All 4 candidates are released 24 hours after a 2Gy radiation dose, which fits with the current clinical dosing schedule where radiation is administered at 24 hour intervals. Ongoing work will elucidate if these biomarkers can be reliably detected in blood or intratumorally using implantable biosensors. · There are currently no validated predictive tools to monitor RT response during treatment. If successfully validated, these biomarkers could have a clinical role in personalising RT dosing schedules and durations for solid tumors in the neoadjuvant and palliative setting, thus optimising treatment and preventing the administration of ineffective RT and its associated side effects. Citation Format: Meehan J, Gray M, Turnbull AK, Martinez-Perez C, Bonello M, Ward C, Langdon SP, McLaughlin S, MacLennan M, Dixon JM, Wills J, Quinn N, Finich AJ, von Kriegsheim A, Cameron D, Kunkler IH, Murray A, Argyle D. Tumor-secreted predictive biomarkers of response to radiotherapy 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 P3-12-24.

Background: Ease of injection is important to patients. An autoinjector was developed to deliver exenatide, a glucagon-like peptide-1 receptor agonist for type 2 diabetes mellitus. For autoinjection, 0.06-mm exenatide-containing microspheres are suspended in medium-chain triglycerides. Herein, we report design verification and usability testing of the autoinjector for exenatide once-weekly suspension (QWS) delivery. Methods: Exenatide QWS in a single-chamber cartridge is self-injected subcutaneously with three main steps: mix, unlock, and inject. Design verification testing used validated testing methodology. A summative validation study with simulated-use scenarios evaluated unassisted performance on critical tasks (ease of use and the injection process). Results: The autoinjector met specified design requirements for dose accuracy and torque/force. Of 104 participants enrolled (73 lay users, 16 health care professionals, and 15 pharmacists), 90 independently referred to instructions for use during testing. Users successfully achieved critical tasks on first attempt 87-100% of the time. Approximately 78% of participants successfully completed the full injection scenario, including 72% of lay users reporting visual or dexterity impairments. Initial use errors on critical tasks included not mixing well (n = 12), not removing needle cap (n = 8), and not holding needle to the skin for complete injection (n = 5). Untrained injection-naïve and trained injection-experienced lay users made the fewest errors (7% and 3%, respectively). Trained and untrained participants took 2:33 and 5:03 minutes, respectively, to complete a weekly injection. Conclusions: Users with a range of injection experience can rapidly learn to administer exenatide QWS autoinjector correctly, thus minimizing patient effort to manage their diabetes with injectable therapy.

The immense costs in both financial terms and preclinical research effort that occur in the development of anticancer drugs are unfortunately not matched by a substantial increase in improved clinical therapies due to the high rate of failure during clinical trials. This may be due to issues with toxicity or lack of clinical effectiveness when the drug is evaluated in patients. Currently, much cancer research is driven by the need to develop therapies that can exploit cancer cell adaptations to conditions in the tumor microenvironment such as acidosis and hypoxia, the requirement for more-specific, targeted treatments, or the exploitation of 'precision medicine' that can target known genomic changes in patient DNA. The high attrition rate for novel anticancer therapies suggests that the preclinical methods used in screening anticancer drugs need improvement. This chapter considers the advantages and disadvantages of 3D organotypic models in both cancer research and cancer drug screening, particularly in the areas of targeted drugs and the exploitation of genomic changes that can be used for therapeutic advantage in precision medicine.

The immense costs in both financial terms and pre-clinical research effort that occur in the development of anti-cancer drugs are unfortunately not matched by a substantial increase in improved clinical therapies due to the high rate of failure during clinical trials. This may be due to issues with toxicity, or lack of clinical ef-fectiveness when the drug is evaluated in patients. Currently, much cancer re-search is driven by the need to develop therapies that can exploit cancer cell ad-aptations to conditions in the tumor microenvironment such as acidosis and hy-poxia, the requirement for more-specific, targeted treatments, or the exploitation of ‘precision medicine’ that can target known genomic changes in patient DNA. The high attrition rate for novel anti-cancer therapies suggests that the pre-clinical methods used in screening anti-cancer drugs need improvement. This chapter considers the advantages and disadvantages of 3D organotypic models in both cancer research and cancer drug screening, particularly in the areas of targeted drugs and the exploitation of genomic changes that can be used for therapeutic advantage in precision medicine

Radiotherapy (RT) is an important treatment modality for the local control of breast cancer (BC). Unfortunately, not all patients that receive RT will obtain a therapeutic benefit, as cancer cells that either possess intrinsic radioresistance or develop resistance during treatment can reduce its efficacy. For RT treatment regimens to become personalised, there is a need to identify biomarkers that can predict and/or monitor a tumour’s response to radiation. Here we describe a novel method to identify such biomarkers. Liquid chromatography-mass spectrometry (LC-MS) was used on conditioned media (CM) samples from a radiosensitive oestrogen receptor positive (ER+) BC cell line (MCF-7) to identify cancer-secreted biomarkers which reflected a response to radiation. A total of 33 radiation-induced secreted proteins that had higher (up to 12-fold) secretion levels at 24 h post-2 Gy radiation were identified. Secretomic results were combined with whole-transcriptome gene expression experiments, using both radiosensitive and radioresistant cells, to identify a signature related to intrinsic radiosensitivity. Gene expression analysis assessing the levels of the 33 proteins showed that 5 (YBX3, EIF4EBP2, DKK1, GNPNAT1 and TK1) had higher expression levels in the radiosensitive cells compared to their radioresistant derivatives, 3 of these proteins (DKK1, GNPNAT1 and TK1) underwent in-lab and initial clinical validation. Western blot analysis using CM samples from cell lines confirmed a significant increase in the release of each candidate biomarker from radiosensitive cells 24 h after treatment with a 2 Gy dose of radiation, no significant increase in secretion was observed in the radioresistant cells after radiation. Immunohistochemistry showed that higher intracellular protein levels of the biomarkers were associated with greater radiosensitivity. Intracellular levels were further assessed in pre-treatment biopsy tissues from patients diagnosed with ER+ BC that were subsequently treated with breast-conserving surgery and RT. High DKK1 and GNPNAT1 intracellular levels were associated with significantly increased recurrence-free survival times, indicating that these two candidate biomarkers have the potential to predict sensitivity to RT. We suggest that the methods highlighted in this study could be utilised for the identification of biomarkers that may have a potential clinical role in personalising and optimising RT dosing regimens, whilst limiting the administration of RT to patients who are unlikely to benefit.

This is a brief memorial tribute to Marshall Berman, which portrays him as a humanist chronicler of the “heroism of modern life.” Berman’s four books achieve this by creative variations in three broad themes: modernism, Marxism, and the metropolis. In Berman’s work, the works of Marx are fruitfully mined for their humanistic vision (with no distinction between the early work and the later “scientific” work), but he makes use of many intellectual and artistic perspectives as well, transcending ideological or disciplinary boundaries, in search of an understanding of the promises and perils of modernism. The metropolis as the theater of the modern life is the implicit or explicit backdrop in his works, and he draws upon his own urban biography to demonstrate the everyday heroism of ordinary people. While vulnerable to criticism as eclectic and naively optimistic, Berman’s writing is critical and empathetic, and true to the spirit of a humanist Marxist understanding of both the disasters and opportunities of modernist capitalist society.

Real‐time monitoring of tumor microenvironment parameters using an implanted biosensor could provide valuable information on the dynamic nature of a tumor's biology and its response to treatment. However, following implantation biosensors may lose functionality due to biofouling caused by the foreign body response (FBR). This study developed a novel tumor xenograft model to evaluate the potential of six biomaterials (silicon dioxide, silicon nitride, Parylene‐C, Nafion, biocompatible EPOTEK epoxy resin, and platinum) to trigger a FBR when implanted into a solid tumor. Biomaterials were chosen based on their use in the construction of a novel biosensor, designed to measure spatial and temporal changes in intra‐tumoral O2, and pH. None of the biomaterials had any detrimental effect on tumor growth or body weight of the murine host. Immunohistochemistry showed no significant changes in tumor necrosis, hypoxic cell number, proliferation, apoptosis, immune cell infiltration, or collagen deposition. The absence of biofouling supports the use of these materials in biosensors; future investigations in preclinical cancer models are required, with a view to eventual applications in humans. To our knowledge this is the first documented investigation of the effects of modern biomaterials, used in the production of implantable sensors, on tumor tissue after implantation. © 2018 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1620–1633, 2019.

Background: A sentinel lymph node in breast cancer surgery is defined as a blue node or a “hot” radioactive node and includes nodes other than the hottest node providing that they contain at least 10% of the radioactivity of the hottest node. The false negative rate of SLNB in breast cancer surgery falls as the number of sentinel nodes removed increases. A recent large US study suggested that patients having 3 or more nodes removed had a better overall survival. Substantial variation remains in how many nodes surgeons remove and what constitutes an adequate SLNB. The aim of this study was to identify what factors influence the number of lymph nodes removed by SNLB. Methods: Data were collected retrospectively from 426 patients with breast cancer who underwent sentinel lymph node biopsy at the Edinburgh Breast Unit by 10 different surgeons between March 2016 and September 2017. Patients with imaging and core biopsy-diagnosed invasive or in situ breast cancer who underwent SLNB as part of breast conserving surgery or mastectomy were eligible. Factors included were patient age, tumour size, tumour grade, type of surgery and surgeon, neoadjuvant chemotherapy (NACT), lympho-vascular invasion, hormone and HER2 receptor status and the number of positive nodes (defined by histological assessment). Univariate and multivariable statistical analyses were performed. Results: The number of sentinel nodes biopsied varied significantly between operating surgeon (p Discussion: • This study shows that the surgeon plays an important and significant role in determining the number of sentinel nodes removed by sentinel lymph node biopsy. • The number of positive nodes was also found to be an important factor, likely due to greater numbers of nodes being taken by surgeons who suspect positive nodes on intraoperatively inspection. • Higher numbers of nodes were removed in younger patients, patients with larger tumours and patients who received NACT. • In conclusion, either some surgeons remove too many nodes or others are removing too few and there needs to be more consistency in surgical SNB practices. Citation Format: J Michael Dixon, Julia Grewar, Dominique Twelves, Charlene Kay, Carlos Martinez-Perez, James Meehan, Mark Gray, Arran K Turnbull. Factors affecting the number of sentinel lymph nodes removed in the treatment and staging of breast cancer? [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-02-09.

Background: 80% of all breast cancers (BCs) are ER+. Not all respond to endocrine therapy (ET) and many eventually develop resistance. An in-depth genomic analysis of cancers that acquired resistance (aR) to multiple sequential lines of ET after an initial period of response has been performed. Patients: A unique cohort of tissues from 20 post-menopausal women with ER+ BC was collected. All patients initially responded to neoadjuvant ET; 19/20 received an aromatase inhibitor (AI): letrozole (LET) (n=18) or anastrozole (AN) (n=1), 1 patient received fulvestrant followed by adjuvant tamoxifen (TAM) (mean treatment duration 22 months, range 4-67) before developing resistance. Patients were then treated by surgery or 2nd line ET. Overall, 13/20 received 2nd line ET either AN (n=1), TAM (n=10) or exemestane (EX) (n=1); 6 went on to receive 3rd line ET with EX (n=5) or LET (n=1). Methods: Serial RNA & DNA from 3-5 cancer samples per patient (89 samples) had Ribo0-RNAseq and DNA exome sequencing. Somatic mutations and copy number alterations (CNA) were determined. Fisher’s exact test was used to compare mutation frequencies (MFs). Differential gene expression analysis was performed using two-class unpaired Significance Analysis of Microarrays (SAM). Results: Mutations: Comparisons of MFs between all sensitive vs resistant tumours identified mutations in SAAL1 and SLC9A9 as being enriched in sensitive tumours while mutations in GATA3 and ZFPM2 were enriched across tumours with acquired resistance to ET. Only KMT2C was identified as enriched in resistant tumours when comparing sensitive to only those that acquired resistance to 1st line LET. When comparing sensitive tumours to only those that had acquired resistance to multiple lines of ET (LET and TAM +/- AN and EX), sensitives were significantly enriched for mutations in CCDC141 and SLC9A9 and multi-drug resistant (MDR) tumours were significantly enriched for mutations in 11 genes including ESR1 and GATA3, and genes involved in cell adhesion (CDH1, FLG, FLG2 and FREM2). ESR1 mutations were identified in 6/20 patients; 5 patients had D538G mutations, 2 appeared during resistance to 1st line LET and 3 in MDR tumours. 1 patient had an ESR1 frame-shift mutation which appeared during resistance to 1st line LET. GATA3 was mutated in 7/20 patients, of which 4 had mutations in all samples including baseline, 2 had mutations uniquely present in MDR tumours and 1 had mutations that appeared during resistance to 1st line LET. CNA: No significant gains or losses were identified when comparing sensitive tumours to those resistant to 1st line LET alone, but significant gains were identified across multiple chromosomes in MDR tumours. Gain of ESR1 was seen in 8/20 patients; in 3 patients there was ESR1 gain at diagnosis which persisted through initial response and subsequent aR. 5 patients developed ESR1 gain during MDR but not during initial resistant to 1st line LET. Gene Expression: Analysis comparing sensitive and MDR tumours identified 82 differentially expressed genes (FDR=0). MDR tumours had higher proliferation and higher expression of transcriptional regulators including FOS and FOSB, histone cluster genes and genes involved in oxidative phosphorylation. MDR tumours also had higher expressions of GATA3 induced genes. Discussion: In contrast to 1st line ET resistance, aR to multiple lines was characterised by significant gains across multiple chromosomes including ESR1 gain in a quarter of tumours. MDR tumours were also characterised by enrichment of mutations in particular genes including ESR1 (D538G in particular) which occurs in one-third of patients with aR. GATA3 is expressed in >90% of BCs, is reported to be mutated in up to 10% of all BCs may be integral to the functions of ESR1; mutations in GATA3 were enriched in MDR tumours and may highlight a new area of ET resistance. Citation Format: Arran K Turnbull, Youli Xa, Carlos Martinez-Perez, Olga Oikonomidou, James Meehan, Mark Gray, Lisa A Carey, Charles M Perou, J Michael Dixon. In-depth genomic analysis of acquired resistance to multiple sequential lines of endocrine therapy in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-03.

The community land trust (CLT) is a social invention designed to solve several problems in land ownership, from affordability to preservation. This article traces the history of the CLT from concept to implementation, through a network of theorists and activists, and discusses the present extent of CLTs in the United States. It concludes with a case study of the Dudley Street Neighborhood Initiative (DSNI), a community development organization in Boston, that has used the CLT model as part of its holistic strategy to redevelop a neighborhood that has suffered from redlining, arson, and abandonment. DSNI is perhaps the only community organization in the United States to have attained the power of eminent domain to acquire land for housing development.

// James Meehan 1 , Carol Ward 1 , Arran Turnbull 1 , Jimi Bukowski-Wills 2 , Andrew J. Finch 2 , Edward J. Jarman 1 , Chrysi Xintaropoulou 1 , Carlos Martinez-Perez 1 , Mark Gray 1 , Matthew Pearson 3 , Peter Mullen 4 , Claudiu T. Supuran 5 , Fabrizio Carta 5 , David J. Harrison 4 , Ian H. Kunkler 1 and Simon P. Langdon 1 1 Cancer Research UK Edinburgh Centre and Division of Pathology Laboratory, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom 2 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom 3 IGMM Advanced Imaging Resource, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom 4 School of Medicine, University of St Andrews, Fife KY16 9TF, United Kingdom 5 Department of Neurofarba, Sez. Chimica Farmaceutica e Nutraceutica, University of Florence, Sesto Fiorentino 50019, Italy Correspondence to: James Meehan, email: J.Meehan@sms.ed.ac.uk Keywords: carbonic anhydrase IX, NHE1, V-ATPase, breast cancer, hypoxia Received: April 14, 2016 Accepted: March 15, 2017 Published: April 17, 2017 ABSTRACT Hypoxic cancer cells exhibit resistance to many therapies. This study compared the therapeutic effect of targeting the pH regulatory proteins (CAIX, NHE1 and V-ATPase) that permit cancer cells to adapt to hypoxic conditions, using both 2D and 3D culture models. Drugs targeting CAIX, NHE1 and V-ATPase exhibited anti-proliferative effects in MCF-7, MDA-MB-231 and HBL-100 breast cancer cell lines in 2D. Protein and gene expression analysis in 2D showed that CAIX was the most hypoxia-inducible protein of the 3 targets. However, the expression of CAIX differed between the 3 cell lines. This difference in CAIX expression in hypoxia was consistent with a varying activity of FIH-1 between the cell lines. 3D expression analysis demonstrated that both CAIX and NHE1 were up-regulated in the hypoxic areas of multicellular tumor spheroids. However, the induction of CAIX expression in hypoxia was again cell line dependent. 3D invasion assays conducted with spheroids showed that CAIX inhibition significantly reduced the invasion of cells. Finally, the capability of both NHE1 and CAIX inhibitors to combine effectively with irradiation was exhibited in clonogenic assays. Proteomic-mass-spectrometric analysis indicated that CAIX inhibition might be combining with irradiation through stimulating apoptotic cell death. Of the three proteins, CAIX represents the target with the most promise for the treatment of breast cancer.

We thank SULSA (Scottish Universities Life Science Alliance) for supporting this project through a SULSA BioSkape Industry PhD Studentship and Antoxis Limited for providing additional funding. We also thank the 7th Framework Programme of the European Union (METOXIA project; HEALTH-F2-2009-222741) for support. Background: The natural polyphenol myricetin induces cell cycle arrest and apoptosis in preclinical cancer models. We hypothesised that myricetin-derived flavonoids with enhanced redox properties, improved cell uptake and mitochondrial targeting might have increased potential as antitumour agents. Methods: We studied the effect of a second-generation flavonoid analogue, Oncamex, in a panel of 7 breast cancer cell lines, applying western blotting, gene expression analysis, fluorescence microscopy, and immunohistochemistry to xenograft tissue to investigate its mechanism of action. Results: Proliferation assays showed that Oncamex: treatment for 8 h reduced cell viability and induced cytotoxicity and apoptosis, concomitant with increased caspase activation. Microarray analysis showed that Oncamex was associated with changes in expression of genes controlling cell cycle and apoptosis . Fluorescence microscopy showed the compound’s mitochondrial targeting and ROS-modulating properties, inducing superoxide production at concentrations associated with anti-proliferative effects. A preliminary in vivo study in mice implanted with the MDA-MB-231 breast cancer xenograft showed that Oncamex inhibited tumour growth, reduced tissue viability and Ki-67 proliferation, with no overall systemic toxicity. Conclusion: Oncamex is a novel flavonoid capable of specific mitochondrial delivery and redox modulation. It has shown antitumour activity in preclinical models of breast cancer, supporting the potential of this prototypic candidate for its continued development as an anticancer agent. Publisher PDF

HAMLET (human α-lactalbumin made lethal to tumour cells) and its related partially unfolded protein–fatty acid complexes are novel biomolecular nanoparticles that possess relatively selective cytotoxic activities towards tumour cells. One of the key characteristics is the requirement for the protein to be partially unfolded, hence endowing native proteins with additional functions in the alternatively folded states. Beginning with the history of its discovery and development, the cellular targets that appear to be strongly correlated with tumour cell death are introduced in the present article.

The role of radiotherapy is well established in the multidisciplinary management of breast cancer. However, its use could be customised further with the intent of enhancing tumour cell radiosensitisation and reducing normal cell toxicity. The importance of tumour heterogeneity and the microenvironment in the response to radiotherapy is under intense scrutiny and the value of molecular profiling is being increasingly recognised. Genome Wide Association Studies are likely toplay an important role in elucidating the molecular pathogenesis of radiotoxicity in the emerging area of radiogenomics. Biomarkers of tumour radiosensitivity should help indicate potentially responsive and unresponsive cancers. Further understanding of the tumour microenvironment and better preclinical models will help identify targets to enhance radiosensitivity or reverse radioresistance.

// Carol Ward 1 , James Meehan 1 , Peter Mullen 2 , Claudiu Supuran 3 , J. Michael Dixon 4 , Jeremy S. Thomas 5 , Jean-Yves Winum 6 , Philippe Lambin 7 , Ludwig Dubois 7 , Nanda-Kumar Pavathaneni 6, 7 , Edward J. Jarman 1 , Lorna Renshaw 4 , InHwa Um 2 , Charlene Kay 1 , David J. Harrison 2 , Ian H. Kunkler 8 , Simon P. Langdon 1 1 Division of Pathology, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom 2 School of Medicine, University of St Andrews, North Haugh, St Andrews, United Kingdom 3 Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Sesto Fiorentino, Florence, Italy 4 Edinburgh Breast Unit, Western General Hospital, Edinburgh, United Kingdom 5 Department of Pathology, Western General Hospital, Edinburgh, United Kingdom 6 Institut des Biomolecules Max Mousseron (IBMM), UMR 5247, CNRS-UM1-UM2, Batiment de Recherche Max Mousseron, Ecole Nationale Superieure de Chimie de Montpellier, Montpellier, France 7 Department of Radiation Oncology (MaastRO), GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands 8 Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom Correspondence to: Carol Ward, e-mail: drcward@hotmail.co.uk Keywords: carbonic anhydrase IX, breast cancer, tumour microenvironment, invasion, hypoxia Received: May 11, 2015 Accepted: June 22, 2015 Published: July 04, 2015 ABSTRACT Triple negative, resistant or metastatic disease are major factors in breast cancer mortality, warranting novel approaches. Carbonic anhydrase IX (CAIX) is implicated in survival, migration and invasion of breast cancer cells and inhibition provides an innovative therapeutic strategy. The efficacy of 5 novel ureido-substituted sulfamate CAIX inhibitors were assessed in increasingly complex breast cancer models, including cell lines in normoxia and hypoxia, 3D spheroids and an ex-vivo explant model utilizing fresh biopsy tissue from different breast cancer subtypes. CAIX expression was evaluated in a tissue microarray (TMA) of 92 paired lymph node and primary breast cancers and 2 inhibitors were appraised in vivo using MDA-MB-231 xenografts. FC11409B, FC9398A, FC9403, FC9396A and S4 decreased cell proliferation and migration and inhibited 3D spheroid invasion. S4, FC9398A and FC9403A inhibited or prevented invasion into collagen. FC9403A significantly reversed established invasion whilst FC9398A and DTP348 reduced xenograft growth. TMA analysis showed increased CAIX expression in triple negative cancers. These data establish CAIX inhibition as a relevant therapeutic goal in breast cancer, targeting the migratory, invasive, and metastatic potential of this disease. The use of biopsy tissue suggests efficacy against breast cancer subtypes, and should provide a useful tool in drug testing against invasive cancers.

Carbonic anhydrase IX has been under intensive investigation as a therapeutic target in cancer. Studies demonstrate that this enzyme has a key role in pH regulation in cancer cells, allowing these cells to adapt to the adverse conditions of the tumour microenviroment. Novel CAIX inhibitors have shown efficacy in bothin vitroandin vivopre-clinical cancer models, adversely affecting cell viability, tumour formation, migration, invasion, and metastatic growth when used alone. In co-treatments, CAIX inhibitors may enhance the effects of anti-angiogenic drugs or chemotherapy agents. Research suggests that these inhibitors may also increase the response of tumours to radiotherapy. Although many of the anti-tumour effects of CAIX inhibition may be dependent on its role in pH regulation, recent work has shown that CAIX interacts with several of the signalling pathways involved in the cellular response to radiation, suggesting that pH-independent mechanisms may also be an important basis of its role in tumour progression. Here, we discuss these pH-independent interactions in the context of the ability of CAIX to modulate the responsiveness of cancer to radiation.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (

e14071Background: Clinical trials now underway will assess the use of flavonoids (natural polyphenols) as anticancer agents. Previous work has shown the promising antitumor activity of Oncamex (a n...

Background: The abnormal regulation of H+ ions, leading to a reversed pH gradient in tumor cells in comparison to normal cells, is considered to be one of the hallmarks of cancer. This feature, however, has yet to be exploited as a therapeutic target. The aim of this study was to assess whether targeting proteins (CAIX, NHE1 and V-ATPase) that permit hypoxic cancer cell adaptation to acidosis in the tumor microenvironment can produce an effective therapeutic response in breast cancer, using 2D and 3D models. Method: Western blotting and gene expression analysis were performed on MCF-7, MDA-MB-231 and HBL-100 cancer cells to assess target protein expression in differing O2 conditions in 2D, while IHC was used to measure protein levels in 3D using multicellular tumor spheroids. Sulforhodamine B assays were executed to analyze the effects of inhibitors targeting CAIX, NHE1 and V-ATPase on breast cancer cell proliferation in 2D. 3D invasion assays were performed with MDA-MB-231 spheroids and explant tissue derived from human patients to see if CAIX inhibition had any effect on cancer cell invasion. An MDA-MB-231 xenograft model was used to investigate the effects of CAIX inhibition in vivo. Clonogenic assays were performed with MDA-MB-231 spheroids to evaluate whether any of the drugs combined effectively with irradiation. Results: 2D and 3D expression analysis showed that CAIX levels were extremely responsive to changes in O2 conditions in each of the cell lines, with HBL100 cells exhibiting the largest changes in both mRNA (42-fold increase) and protein (78-fold increase) levels at low (0.5%) O2 concentrations. NHE1 and V-ATPase mRNA/protein levels were, however, much more consistently expressed across the cell lines in different O2 conditions. Drugs targeting CAIX, NHE1 and V-ATPase had anti-proliferative effects on the breast cancer cells in 2D. Normoxic cancer cells were the most sensitive to drug treatment, acute hypoxic cancer cells showed increased resistance to the anti-proliferative effects of these drugs, while chronic hypoxic cells had IC50 values more similar to the normoxic cells. The results for the CAIX inhibitor were unexpected, as we had predicted that the increased levels of CAIX in the acute hypoxic cells would make them more sensitive to treatment. CAIX inhibition did, however, significantly reduce the invasion of cancer cells from both MDA-MB-231 spheroids (p≤0.01) and explant tissue (p≤0.001). Targeting pH regulation was also shown to have an effect in vivo on MDA-MB-231 xenografts, with CAIX inhibition significantly reducing the growth (p≤0.05) and proliferation (p≤0.05) of tumors within mice. Finally, clonogenic assays showed that drugs targeting both CAIX and NHE1 led to a significant reduction in colony number when combined with radiation (p≤0.05), compared to either drug individually or radiation treatment alone. Conclusions: This study shows that drugs targeting pH regulation molecules have potential in the treatment of breast cancer. This is highlighted by their ability to affect the proliferation and invasion of breast cancer cells, along with their ability to be combined with radiation. Of the 3 pH regulatory molecules, CAIX represents the target with the most promise. Citation Format: Meehan J, Ward C, Jarman E, Xintaropoulou C, Martinez-Perez C, Turnbull A, Supuran C, Dixon M, Kunkler I, Langdon SP. Targeting the pH regulatory mechanisms of breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-05.

We recently reported that mice treated with 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) or 19-nor-1,25-(OH) 2 D 2 experienced a severe loss of their thymocytes and decreased proliferation in response to concanavalin A mitogen. The present study investigated the effect of short-term treatment with 1,25-(OH) 2 D 3 on the thymic architecture and thymocyte subsets. Daily treatment with 1,25-dihydroxyvitamin D 3 at 20 ng permouse for 4 days induced significant involution of thymic tissue. The atrophy was predominantly observed in the cortical component. Flow cytometric analysis of thymocyte subsets showed that the CD4 + CD8 + population was the primary target. Since the treated mice experienced profound hypercalcemia, we studied the effect of 1,25-(OH) 2 D 3 on animals fed a vitamin D-deficient, low calcium diet or the same diet containing vitamin D for 25 days prior to treatment. The low calcium fed mice showed severe hypocalcemia and slight thinning of thymic cortex. Treatment with 1,25-(OH) 2 D 3 moderately improved the hypocalcemia but had no further effect on the thymus of these animals. On the other hand, hypercalcemia and thymic atrophy were found in the animals fed the diet containing vitamin D. Overall, the atrophy effect on the thymus caused by 1,25-(OH) 2 D 3 treatment was prevented by eliminating the hypercalcemia observed in + D + Ca treated animals. Thus, thymic atrophy probably resulted from hypercalcemia and not from 1,25-(OH) 2 D 3 itself.