BRCA1-like signature in triple negative breast cancer: Molecular and clinical characterization reveals subgroups with therapeutic potential.

Triple negative (TN) breast cancers make up some 15% of all breast cancers. Approximately 10-15% are mutant for the tumor suppressor, BRCA1. BRCA1 is required for homologous recombination-mediated DNA repair and deficiency results in genomic instability. BRCA1-mutated tumors have a specific pattern of genomic copy number aberrations that can be used to classify tumors as BRCA1-like or non-BRCA1-like. BRCA1 mutation, promoter methylation, BRCA1-like status and genome-wide expression data was determined for 112 TN breast cancer samples with long-term follow-up. Mutation status for 21 known DNA repair genes and PIK3CA was assessed. Gene expression and mutation frequency in BRCA1-like and non-BRCA1-like tumors were compared. Multivariate survival analysis was performed using the Cox proportional hazards model. BRCA1 germline mutation was identified in 10% of patients and 15% of tumors were BRCA1 promoter methylated. Fifty-five percent of tumors classified as BRCA1-like. The functions of genes significantly up-regulated in BRCA1-like tumors included cell cycle and DNA recombination and repair. TP53 was found to be frequently mutated in BRCA1-like (P < 0.05), while PIK3CA was frequently mutated in non-BRCA1-like tumors (P < 0.05). A significant association with worse prognosis was evident for patients with BRCA1-like tumors (adjusted HR = 3.32, 95% CI = 1.30-8.48, P = 0.01). TN tumors can be further divided into two major subgroups, BRCA1-like and non-BRCA1-like with different mutation and expression patterns and prognoses. Based on these molecular patterns, subgroups may be more sensitive to specific targeted agents such as PI3K or PARP inhibitors

The BRCA1ness signature is associated significantly with response to PARP inhibitor treatment versus control in the I-SPY 2 randomized neoadjuvant setting.

BACKGROUND: Patients with BRCA1-like tumors correlate with improved response to DNA double-strand break-inducing therapy. A gene expression-based classifier was developed to distinguish between BRCA1-like and non-BRCA1-like tumors. We hypothesized that these tumors may also be more sensitive to PARP inhibitors than standard treatments. METHODS: A diagnostic gene expression signature (BRCA1ness) was developed using a centroid model with 128 triple-negative breast cancer samples from the EU FP7 RATHER project. This BRCA1ness signature was then tested in HER2-negative patients (n = 116) from the I-SPY 2 TRIAL who received an oral PARP inhibitor veliparib in combination with carboplatin (V-C), or standard chemotherapy alone. We assessed the association between BRCA1ness and pathologic complete response in the V-C and control arms alone using Fisher's exact test, and the relative performance between arms (biomarker × treatment interaction, likelihood ratio p < 0.05) using a logistic model and adjusting for hormone receptor status (HR). RESULTS: We developed a gene expression signature to identify BRCA1-like status. In the I-SPY 2 neoadjuvant setting the BRCA1ness signature associated significantly with response to V-C (p = 0.03), but not in the control arm (p = 0.45). We identified a significant interaction between BRCA1ness and V-C (p = 0.023) after correcting for HR. CONCLUSIONS: A genomic-based BRCA1-like signature was successfully translated to an expression-based signature (BRC1Aness). In the I-SPY 2 neoadjuvant setting, we determined that the BRCA1ness signature is capable of predicting benefit of V-C added to standard chemotherapy compared to standard chemotherapy alone. TRIAL REGISTRATION: I-SPY 2 TRIAL beginning December 31, 2009: Neoadjuvant and Personalized Adaptive Novel Agents to Treat Breast Cancer (I-SPY 2), NCT01042379

Pitfalls in assessing stromal tumor infiltrating lymphocytes (sTILs) in breast cancer

Abstract: Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls

Report on computational assessment of Tumor Infiltrating Lymphocytes from the International Immuno-Oncology Biomarker Working Group

Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)Funder: National Center for Research Resources under award number 1 C06 RR12463-01, VA Merit Review Award IBX004121A from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, the DOD Prostate Cancer Idea Development Award (W81XWH-15-1-0558), the DOD Lung Cancer Investigator-Initiated Translational Research Award (W81XWH-18-1-0440), the DOD Peer Reviewed Cancer Research Program (W81XWH-16-1-0329), the Ohio Third Frontier Technology Validation Fund, the Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering and the Clinical and Translational Science Award Program (CTSA) at Case Western Reserve University.Funder: Susan G Komen Foundation (CCR CCR18547966) and a Young Investigator Grant from the Breast Cancer Alliance.Funder: The Canadian Cancer SocietyFunder: Breast Cancer Research Foundation (BCRF), Grant No. 17-194Abstract: Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring

Application of a risk-management framework for integration of stromal tumor-infiltrating lymphocytes in clinical trials

Funder: Breast Cancer Research Foundation (BCRF); doi: https://doi.org/10.13039/100001006Abstract: Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting

Effets de l'amantadine sur la transmission dopaminergique : rôle du récepteur sigma1 = Effects of amantadine on dopaminergic transmission. Implication of the sigma1 receptor

Parkinson's disease affects approximately one of every 1000 persons over the world. It constitutes the most common neurodegenerative disorder of the elderly after the Alzheimer's disease. Usually associated with loss of dopaminergic nigral neurons and depletion of dopamine levels in both substantia nigra and striatum, Parkinson's disease, unlike most of neurodegenerative diseases, may be accessible to drug therapy. Amantadine, empirically used for more than 30 years to alleviate locomotor symptoms in Parkinsons disease, has recently been found to reduce dyskinesias induced by chronic L-DOPA therapy. This non competitive NMDA glutamate receptor antagonist is proposed to exert its therapeutic effects by increasing the dopaminergic transmission. Nevertheless, less is known about the biochemical mechanisms involved in this modulation of dopaminergic pathway. This work aims to study the effects of repeated administrations of amantadine on dopaminergic transmission, in particular on the expression and on the functional activity of dopamine receptors and transporter. Our results show that a 4-day amantadine treatment enhances the functional coupling between dopamine receptors and G-protein without any changes of dopamine receptor density. These biochemical data correlate with behavioural analyse as a sensitisation of rats behavioural response after the administration of a dopaminergic agonist (apomorphine) was also observed. In order to determine if this response was sustained or not, the effects of a longer treatment (7 days) were studied revealing the development of compensatory mechanisms including a decrease in dopamine receptor density and an enhancement of dopamine transporter activity. The implication of a second molecular target for amantadine, the sigma1 receptor, was then suggested. First, the pharmacological profile of amantadine for the sigma1 receptor was determined. Then, the sensitisation of dopamine transmission induced by repeated amantadine administrations was demonstrated to reflect the interaction of amantadine with the sigma1 receptor. As the activation of this receptor is involved in the regulation of membrane fluidity and cytoskeletal protein activity, this may constitute a relevant explanation for the modulation of G-protein activity observed after amantadine administrations. These works lead to the identification of a new potential molecular target of amantadine which could be involved in the regulation of the functional activity of dopamine receptors. They may also contribute to the development of new therapeutic strategies in the treatment of Parkinson's disease.La maladie de Parkinson atteint environ une personne sur mille dans le monde. Elle représente l'affection neurologique dégénérative liée à l'âge la plus fréquente après la maladie d'Alzheimer. Habituellement associée à une perte des neurones dopaminergiques de la substance noire et une diminution de la concentration de dopamine, à la fois dans la substance noire et le striatum, la maladie de Parkinson est l'une des rares atteintes neurodégénératives accessible à la thérapeutique médicamenteuse. L'amantadine, molécule utilisée de manière empirique depuis plus de 30 ans dans le traitement des symptômes locomoteurs de la maladie de Parkinson, connaît à l'heure actuelle un regain d'intérêt pour le traitement des dyskinésies tardives induites par la L-DOPA. Les effets bénéfiques de cette molécule résulteraient d'une régulation positive de la transmission dopaminergique induite par son activité en tant qu'antagoniste non compétitif du récepteur glutamatergique de type NMDA. Néanmoins, à l'heure actuelle, les mécanismes neurochimiques sous-jacents à ses propriétés thérapeutiques sont encore mal compris. Ce travail a pour but l'étude des effets d'administrations répétées d'amantadine sur la transmission dopaminergique et, en particulier, sur l'expression et l'activité fonctionnelle des récepteurs et du transporteur de la dopamine. Nos résultats indiquent que l'administration répétée d'amantadine pendant 4 jours améliore l'efficacité de couplage des récepteurs dopaminergiques aux protéines G sans modifier le nombre de récepteurs dopaminergiques. Ces données biochimiques sont confirmées par une sensibilisation de la réponse comportementale induite par un agoniste dopaminergique (l'apomorphine) chez des rats traités avec l'amantadine. L'administration d'amantadine sur une plus longue période (7 jours) révèle le caractère transitoire de la réponse et la mise en route de mécanismes compensatoires, tels une diminution de la densité des récepteurs dopaminergiques et une augmentation de l'activité du transporteur neuronal de la dopamine. L'interaction de l'amantadine, aux concentrations thérapeutiques, avec une seconde cible moléculaire, le récepteur sigma1, a alors été suggérée. Après une caractérisation du profil pharmacologique de l'amantadine pour le récepteur sigma1, l'implication de l'activation de ce dernier dans la sensibilisation de la transmission dopaminergique induite par des administrations répétées d'amantadine a été démontrée.L'activation de ce récepteur, impliqué dans la régulation de la fluidité membranaire et de l'activité de protéines du cytosquelette, pourrait expliquer l'effet d'administrations répétées d'amantadine sur la modulation de l'activité des protéines G. Ces travaux ont permis d'identifier une nouvelle cible moléculaire de l'amantadine, laquelle pourrait être impliquée dans la régulation de l'activité fonctionnelle des récepteurs dopaminergiques. Ils pourront également contribuer au développement de nouvelles approches thérapeutiques pour le traitement de la maladie de Parkinson.Thèse de doctorat en sciences pharmaceutiques (FARM 3) -- UCL, 200

Distinct effects of amantadine and memantine on dopaminergic transmission in the rat striatum.

Striatal glutamatergic inputs are known to participate in the modulation of dopaminergic transmission. Accordingly, the non-competitive N-methyl-D-aspartate receptor antagonists memantine and amantadine increase striatal dopamine levels, the latter being widely used in Parkinson's disease therapy. Based on our previous work revealing increased function of dopamine receptors and dopamine transporter after amantadine treatment, we studied the effects of repeated memantine administration on dopaminergic neurotransmission. On rat striatal membranes, dopamine-stimulated [(35)S]GTPgammaS binding was significantly reduced (20%) after 2 days injection with memantine (20 mg/kg per day, i.p.) but not after longer treatments (4 or 7 days). Evaluation of [(3)H]SCH 23390 and [(3)H]spiperone specific bindings only revealed a significant increase in D1 receptor density after 4 or 7 days treatment. Finally, none of these treatments were found to change the activity of the neuronal dopamine transporter in striatal synaptosomes. This shows that amantadine and memantine differentially affect striatal dopaminergic transmission, which could indicate that these two related aminoadamantanes display distinct pharmacodynamic properties

Increased dopamine uptake in striatal synaptosomes after treatment of rats with amantadine.

The aim of the present study was to investigate the effect of short- and long-term treatments with amantadine on the activity of the neuronal dopamine transporter (DAT) in the rat striatum. For this purpose, the [3H]dopamine uptake was measured in striatal synaptosomes prepared from rats treated for 2, 7 and 14 days with amantadine (40 mg/kg; i.p.). After 7 days of treatment, amantadine increased the apparent V(max) by 30% without modification of the apparent K(m) of dopamine uptake whereas no change in these parameters was observed after 2 and 14 days treatment. Binding assays conducted with [3H]GBR-12935 on membranes prepared from animals treated with amantadine revealed no difference in the density and the affinity of striatal DAT binding sites as compared to control. This indicates that the increased dopamine uptake was not reflecting a modification at the level of the DAT expression. The activity of the DAT is regulated by phosphorylation and one may propose that ionotropic glutamate receptors present on presynaptic terminals directly modulate this phosphorylation. An indirect mechanism would involve presynaptic dopamine receptors that control the activity of the DAT in response to the increased dopamine concentration in the synaptic cleft

Hypersensitivity of dopamine transmission in the rat striatum after treatment with the NMDA receptor antagonist amantadine.

Amantadine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist known to increase dopamine synthesis and release in the striatum, is frequently associated with L-DOPA in the treatment of Parkinson's disease. However, the biochemical mechanisms involved in the effect of amantadine and the consequences of its repetitive administration on the modulation of striatal dopamine transmission still need to be clarified. We have investigated the effects of short-term amantadine treatments on the expression of dopamine receptors and the functional coupling to G proteins in rat striatal membranes. Dopamine-induced stimulation of guanosine 5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding was significantly enhanced (40%) in striatum homogenates from rats treated for 4 days with amantadine (40 mg/kg, i.p.) compared to vehicle-treated animals. This effect was specific for dopamine receptors and was transient as no significant modifications were observed when animals were treated for either 2 or 7 days. Administration of amantadine did not directly affect the animal behaviour. However, treated animals exhibited hypersensitive dopamine transmission since rats treated for 4 days showed exacerbated responses to a single apomorphine administration (enhanced locomotor activity and reduced stereotypy). Since the effects of amantadine administration differ from those usually observed with direct dopamine receptor agonists or other NMDA receptor antagonists, we suggest that multiple biochemical mechanisms contribute to the modulation of dopamine transmission by amantadine