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Among others, expression levels of programmed cell death 1 ligand 1 (PD‐L1) have been explored as biomarkers of the response to immune checkpoint inhibitors in cancer therapy. Here, we present the results of a chemical screen that interrogated how medically approved drugs influence PD‐L1 expression. As expected, corticosteroids and inhibitors of Janus kinases were among the top PD‐L1 downregulators. In addition, we identified that PD‐L1 expression is induced by antiestrogenic compounds. Transcriptomic analyses indicate that chronic estrogen receptor alpha (ERα) inhibition triggers a broad immunosuppressive program in ER‐positive breast cancer cells, which is subsequent to their growth arrest and involves the activation of multiple immune checkpoints together with the silencing of the antigen‐presenting machinery. Accordingly, estrogen‐deprived MCF7 cells are resistant to T‐cell‐mediated cell killing, in a manner that is independent of PD‐L1, but which is reverted by estradiol. Our study reveals that while antiestrogen therapies efficiently limit the growth of ER‐positive breast cancer cells, they concomitantly trigger a transcriptional program that favors their immune evasion., Under prolonged hormone therapy, ER+ breast cancer cells activate an inflammatory transcriptional program, which includes a generalized upregulation of immune checkpoint mediators together with the downregulation of the antigen‐presenting machinery. These findings reveal that, while hormone therapies efficiently arrest the growth of ER+ breast cancer cells, they also promote a phenotype switch that favors their immune evasion.

Purpose: We previously demonstrated that mitochondrial inhibitors' efficacy was restricted to a metabolic context in which mitochondrial respiration was the predominant energy source, a situation achievable by inducing vascular normalization/hypoxia correction with antiangiogenics. Vascular normalization can be tracked with 2[18F]fluoro-2-deoxy-d-glucose (FDG)-PET. We tested the efficacy of the mitochondrial inhibitor ME-344 or placebo added to bevacizumab in early breast cancer. Patients and Methods: Treatment-naïve HER2-negative patients with T > 1 cm (any N) underwent a breast-centered 18F-fluorodeoxyglucose (FDG)-PET (day 1) and received a single dose of bevacizumab (15 mg/kg), followed by a second FDG-PET (day 8). Patients were then randomized (1:1) to Arm A (ME-344 10 mg/kg intravenous on days 8, 15, and 21) or Arm B (placebo). Tumors were biopsied on days 0 and 29. Succinate dehydrogenase enzyme histochemistry (SDH-EHC), confocal microscopy of vessel architecture, and HIF1α staining were performed in pre- and posttreatment biopsies to assess the pharmacodynamics, vessel normalization, and tissue re-oxygenation by bevacizumab, respectively. Results: ME-344 displayed significant biological activity versus placebo: compared with a 186% increase in Arm B, Ki67 decreased by 23.4% from days 0 to 28 in Arm A (P < 0.001) (N = 42 patients). FDG-PET predicted vascular normalization in about one-third of the patients in each arm, which was confirmed using confocal microscopy and HIF1α staining. In the subgroup with vascular normalization, ME-344 induced a Ki67 decrease of 33.4% (placebo: 11.8 increase). SDH-EHC suggested on-target effects of ME-344. Conclusions: ME-344 has significant biological antitumor activity in HER2-negative breast cancer, particularly after induction of vascular normalization and tissue reoxygenation with bevacizumab.

Simple Summary Triple-negative breast cancers (TNBC) represent approximately 15% of all breast cancers and lack the expression of a defined molecular target. This absence makes this subtype of cancer difficult to treat and control. Current chemotherapy drugs cause various side effects and toxicities that can jeopardize the quality of life of patients with TNBC cancer. Therefore, this research focuses on a new semi-synthetic compound derived from [6]-gingerol, where we demonstrate that it does not cause significant toxic effects in vivo and, more importantly, we demonstrate its antitumor and antimetastatic effects using preclinical xenograft models simulating two clinical scenarios of a woman with breast cancer. Abstract Breast cancer metastasis is the most common cause of cancer death in women worldwide. Triple-negative breast cancers (TNBC) form a heterogeneous group of tumors that have higher relapse rates and poorer survival compared to other breast cancer subtypes. Thus, this work reports the antitumor and antimetastatic activities of a [6]-gingerol-derived semi-synthetic compound named SSi6 on MDA-MB-231 TNBC cells using xenograft models. SSi6 did not cause toxic effects in vivo as demonstrated by body weight and hematological and histological evaluations. From the orthotopic xenograft model, we demonstrated that SSi6 slows and inhibits the growth of the primary tumor, as well as prevents metastatic spontaneous progression from lymph nodes to the lungs. Moreover, a second xenograft model with resection of the primary tumor showed that SSi6 also blocks the progression of metastases from the lymph nodes to other visceral organs. Taken together, our results demonstrate that SSi6 is a promising compound to be investigated in other preclinical and clinical models to be applied as a complementary therapy for TNBC.

Preclinical evidence indicates the potential of targeting mitochondrial respiration as a therapeutic strategy. We previously demonstrated that mitochondrial inhibitors' efficacy was restricted to a metabolic context in which mitochondrial respiration was the predominant energy source, a situation achievable by inducing vascular normalization/hypoxia correction with antiangiogenics. Using molecular imaging, we showed how the same antiangiogenic agent may display different normalizing properties in patients with the same tumor type. This is of key importance, since patients experiencing normalization seem to get more benefit from standard chemotherapy combinations, and also could be eligible for combination with antimitochondrial agents. This scenario emphasizes the need for monitoring vascular normalization in order to optimize the use of antiangiogenics. We have also proposed a method to evaluate anti-mitochondrial agents' pharmacodynamics; despite promising accuracy in animal studies the clinical results were inconclusive, highlighting the need for research in this field. Regarding patients that respond to antiangiogenics increasing vessel abnormality, in this case an immunosuppressive tumor microenvironment is generated. Whether anti-mitochondrial agents can positively modulate the activity of T effector cell subpopulations remains an area of active research. Our research sheds light on the importance of refining the use of antiangiogenics, highlighting the relevance of tracing vascular normalization as a potential biomarker for antiangiogenics to assist patient-tailored medicine and exploring the role of mitochondrial inhibitors in the context of vascular normalization and correction of hypoxia. Sí

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations, Background FGFR1 amplification, but not overexpression, has been related to adverse prognosis in hormone-positive breast cancer (HRPBC). Whether FGFR1 overexpression and amplification are correlated, what is their distribution among luminal A or B HRPBC, and if there is a potential different prognostic role for amplification and overexpression are currently unknown features. The role of FGFR1 inhibitors in HRPBC is also unclear. Methods FGFR1 amplification (FISH) and overexpression (RNAscope) were investigated in a N = 251 HRPBC patients cohort and the METABRIC cohort; effects on survival and FISH-RNAscope concordance were determined. We generated hormonal deprivation resistant (LTED-R) and FGFR1-overexpressing cell line variants of the ER+ MCF7 and T47-D and the ER+, FGFR1-amplified HCC1428 cell lines. The role of ER, CDK4/6, and/or FGFR1 blockade alone or in combinations in Rb phosphorylation, cell cycle, and survival were studied. Results FGFR1 overexpression and amplification was non-concordant in > 20% of the patients, but both were associated to a similar relapse risk (~ 2.5-fold; P, MQF is a recipient of the following grants: AES - PI16/00354 and AES – PI19/00454 funded by the ISCIII (Instituto de Salud Carlos III, Ministry of Science and Innovation, Spain) and co-funded by the European Regional Development Fund (ERDF); 2017/BMD3733 - Call for Coordinated Research Groups from Madrid Region - Madrid Regional Government - ERDF funds. RC and MJB are co-recipients of an AES – PI17/01865 grant funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF). RC is a recipient of funding PIE15/00068, PI20/01458, and PT17/0017/0007 by the ISCIII. This study was partially funded by Bayer Inc. CRIS Contra el Cancer Foundation contributed with a generous donation to this study

© The Author(s)., [Background]: Preclinical research suggests that the efficacy of immune checkpoint inhibitors in breast cancer can be enhanced by combining them with antiangiogenics, particularly in a sequential fashion. We sought to explore the efficacy and biomarkers of combining the anti-PD-L1 durvalumab plus the antiangiogenic bevacizumab after bevacizumab monotherapy for advanced HER2-negative breast cancer. [Methods]: Patients had advanced HER2-negative disease that progressed while receiving single-agent bevacizumab maintenance as a part of a previous chemotherapy plus bevacizumab regimen. Treatment consisted of bi-weekly durvalumab plus bevacizumab (10 mg/kg each i.v.). Peripheral-blood mononuclear cells (PBMCs) were obtained before the first durvalumab dose and every 4 weeks and immunophenotyped by flow-cytometry. A fresh pre-durvalumab tumor biopsy was obtained; gene-expression studies and immunohistochemical staining to assess vascular normalization and characterize the immune infiltrate were conducted. Patients were classified as “non-progressors” if they had clinical benefit (SD/PR/CR) at 4 months. The co-primary endpoints were the changes in the percentage T cell subpopulations in PBMCs in progressors versus non-progressors, and PFS/OS time. [Results]: Twenty-six patients were accrued. Median PFS and OS were 3.5 and 11 months; a trend for a longer OS was detected for the hormone-positive subset (19.8 versus 7.4 months in triple-negatives; P = 0.11). Clinical benefit rate at 2 and 4 months was 60% and 44%, respectively, without significant differences between hormone-positive and triple-negative (P = 0.73). Non-progressors’ tumors displayed vascular normalization features as a result of previous bevacizumab, compared with generally abnormal patterns observed in progressors. Non-progressors also showed increased T-effector and T-memory signatures and decreased TREG signatures in gene expression studies in baseline—post-bevacizumab—tumors compared with progressors. Notably, analysis of PBMC populations before durvalumab treatment was concordant with the findings in tumor samples and showed a decreased percentage of circulating TREGs in non-progressors. [Conclusions]: This study reporting on sequential bevacizumab+durvalumab in breast cancer showed encouraging activity in a heavily pre-treated cohort. The correlative studies agree with the preclinical rationale supporting an immunopriming effect exerted by antiangiogenic treatment, probably by reducing TREGs cells both systemically and in tumor tissue. The magnitude of this benefit should be addressed in a randomized setting., MQF is a recipient of the following grants: AES - PI16/00354 funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) and B2017/BMD3733 (Immunothercan-CM) - Call for Coordinated Research Groups from Madrid Region - Madrid Regional Government - ERDF funds. SM is a recipient of the following grants: Spanish Ministerio de Economía y Competitividad (MINECO) (SAF2017-83732-R; AEI/FEDER, EU) and co-funded by Comunidad de Madrid (B2017/BMD3733; Immunothercan-CM). RC is a recipient RC is a recipient of the ISCIII grants PIE15/00068 and PI17/01865.

Estrogen receptor (ER)-positive breast tumors are routinely treated with estrogen-depriving therapies. Despite their effectiveness, patients often progress into a more aggressive form of the disease. Through a chemical screen oriented to identify chemicals capable of inducing the expression of the immune-checkpoint ligand PD-L1, we found antiestrogens as hits. Subsequent validations confirmed that estrogen deprivation or ERα depletion induces PD-L1 expression in ER-positive breast cancer cells, both in vitro and in vivo. Likewise, PD-L1 expression is increased in metastasis arising from breast cancer patients receiving adjuvant hormonal therapy for their local disease. Transcriptome analyses indicate that estrogen deprivation triggers a broad immunosuppressive program, not restricted to PD-L1. Accordingly, estrogen deprived MCF7 cells are resistant to T-cell mediated cell killing, in a manner that can be reverted by estradiol. Our study reveals that while antiestrogen therapies effectively limit tumor growth in ER-positive breast cancers, they also trigger a transcriptional program that favors immune evasion.

The combined use of a FGFR1 blocker and aromatase inhibitors is appealing for treating breast cancer patients with FGFR1 amplification. However, no pharmacodynamic studies have addressed the effects of this combined target modulation. We conducted a phase 0/I clinical trial in an adjuvant setting, with the goal of obtaining pharmacodynamic proof of the effects of combined aromatase and FGFR1 inhibition and to establish the RP2D for nintedanib combined with letrozole. Patients and methods: Women with early-stage luminal breast cancer were eligible for enrollment in the study. Dose level 1 was nintedanib (150 mg/bid) plus letrozole (2.5 mg/day) administered for a single 28-day cycle (DLT assessment period), followed by a classic 3 + 3 schedule. FGF23 and 17-B-estradiol levels were determined on days 0 and 15; pharmacokinetic parameters were assessed on days 1 and 28. Patients were allowed to continue treatment for 6 cycles. The primary study endpoint was a demonstration of FGFR1 modulation (defined as a 25% increase in the plasma FGF23 level). Results: A total of 19 patients were enrolled in the study (10 in the expansion cohort following dose escalation). At the RP2D (nintedanib 200 mg/bid plus letrozole 2.5 mg/day), we observed a 55% mean increase in the plasma FGF23 level, and 81.2% of the patients had no detectable level of 17-B-estradiol in their plasma (87.5% of the patients treated with letrozole alone). Nintedanib and letrozole displayed a pharmacokinetic interaction that led to three- and twofold increases in their respective plasma concentrations. Most G3 toxic events (5 out of 6: 2 diarrhea and 3 hypertransaminasemia) occurred subsequent to the DLT assessment period. Conclusion: Combined treatment with nintedanib (200 mg/bid) plus letrozole (2.5 mg/day) effectively suppressed FGFR1 and aromatase activity, and these respective doses can be used as starting doses in any subsequent trials. However, drug-drug interactions may produce tolerability issues when these drugs are co-administered for an extended time period (e.g., 6 months). Patients enrolled in future trials with these drugs should be carefully monitored for their FGF23 levels and signs of toxicity, and those findings should guide individualized treatment decisions, MQF is a recipient of the following grants: AES - PI16/00354 funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) and B2017/BMD3733 - Call for Coordinated Research Groups from Madrid Region - Madrid Regional Government - ERDF funds. RC is a recipient of the following grants: AES PI17/01865 and PIE15/00068 by the ISCIII and co-funded by the European Regional Development Fund (ERDF). This study was partially funded by Boehringer-Ingelheim. CRIS Contra el Cancer Foundation contributed with a generous donation to this study

3100 Background: We have shown that when Aas induce vascular normalization (VN), tumors escape upregulating mitochondrial metabolism. Mitochondrial inhibition with ME344 induced synergy with various Aas. We also found that VN could be traced by showing a 10% decrease in tumor FDG-PET SUV from day (d) 0 to d8 of Aa. We studied the activity of adding ME344 or placebo to Bev (Ki67 decrease) in E-HERNEBC in a phase 0 randomized trial. As a secondary objective we measured the activity of the combination in patients (Pts) showing VN according to FDG-PET. Methods: Untreated E-HERNEBC Pts with T > 1cm, any N, M0 underwent a baseline FDG-PET (d1) and received a single dose of Bev (15mg/kg) prior to randomization (1:1) to arm A (FDG-PET on d8 followed by ME344 10 mg/kg IV on d8, d15 and d21) or Arm B (FDG-PET on d8 followed by placebo on d8, d15 and d21). Tumors were biopsied on d0 and 28. A 40 Pts sample size was powered to detect a 30% relative difference between arms in digitally acquired Ki67 decrease from d0 to d28 (alpha 0.05, beta 0.2). Results: Arm A: 20 Pts; Arm B: 21 Pts. Baseline characteristics were in arm A vs B: age 58.4(41.5-75.3) vs 53.6(39-82.8); T1(30%)/T2(60%)/T3(10%) vs T1(52%)/T2(48%)/T3(0%); N0(80%)/N1(20%) vs N0(81%)/N1(19%); ER+(75%)/TNBC(25%) vs ER+(71.4%)/TNBC(28.6%); Ki67 31.6% (3.6%-70%) vs 25.2% (1.2% - 81.5%). PET-SUV decreased > 10% from d0 to d8 in 6/20 (arm A) and 6/21 (arm B) Pts. Two G3 adverse events (blood pressure) were reported (1/arm) and deemed related to Bev. Results of the primary endpoint: table. Conclusions: ME344 showed significant biologic activity, enhancing the effect in Ki67 decrease vs placebo when added to Bev in E-HERNEBC. The activity was greater in TNBC. A trend for greater activity in patients experiencing VN according to FDG-PET was observed. Clinical trial information: NCT02806817. [Table: see text]

// Miguel Quintela-Fandino 1 , Nora Soberon 2 , Ana Lluch 3 , Luis Manso 4 , Isabel Calvo 5 , Javier Cortes 6, 7 , Fernando Moreno-Anton 8 , Miguel Gil-Gil 9 , Noelia Martinez-Janez 7 , Antonio Gonzalez-Martin 10 , Encarna Adrover 11 , Raquel de Andres 12 , Gemma Vinas 13 , Antonio Llombart-Cussac 14 , Emilio Alba 15 , Silvana Mouron 1 , Juan Guerra 16 , Begona Bermejo 3 , Esther Zamora 6 , Jose Angel Garcia-Saenz 8 , Sonia Pernas Simon 9 , Eva Carrasco 17 , Maria Jose Escudero 17 , Ruth Campo 17 , Ramon Colomer 18 , Maria A. Blasco 2 1 Breast Cancer Clinical Research Unit, CNIO-Spanish National Cancer Research Center, Madrid, Spain 2 Telomeres and Telomerase Group, Molecular Oncology Programme, CNIO-Spanish National Cancer Research Center, Madrid, Spain 3 Medical Oncology Department, Hospital Clinico Universitario, Valencia, Spain 4 Medical Oncology Department, Hospital 12 de Octubre, Madrid, Spain 5 Medical Oncology Department, Hospital de Monteprincipe, Madrid, Spain 6 Medical Oncology Department, Hospital Vall d´Hebron, Barcelona, Spain 7 Medical Oncology Department, Hospital Ramon y Cajal, Madrid, Spain 8 Medical Oncology Department, Hospital Clinico San Carlos, Madrid, Spain 9 Medical Oncology Department, Institut Catala d'Oncologia-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain 10 Medical Oncology Department, MD Anderson Cancer Center, Madrid, Spain 11 Medical Oncology Department, Hospital General de Albacete, Albacete, Spain 12 Medical Oncology Department, Hospital Lozano Blesa, Zaragoza, Spain 13 Medical Oncology Department, Institut Catala d'Oncologia-Hospital Dr. Josep Trueta, Girona, Spain 14 Medical Oncology Department, Hospital Arnau de Vilanova, Valencia, Spain 15 Medical Oncology Department, Hospital Clinico Universitario Virgen de la Victoria, Malaga, Spain 16 Medical Oncology Department, Hospital Universitario de Fuenlabrada, Fuenlabrada, Spain 17 GEICAM, Madrid, Spain 18 Medical Oncology Department, Hospital Universitario La Princesa, Madrid, Spain Correspondence to: Miguel Quintela-Fandino, email: mquintela@cnio.es Maria A. Blasco, email: mblasco@cnio.es Keywords: telomere length, critically short telomeres, breast cancer, weekly paclitaxel, toxicity Received: October 25, 2016 Accepted: January 27, 2017 Published: February 21, 2017 ABSTRACT Cumulative toxicity from weekly paclitaxel (myalgia, peripheral neuropathy, fatigue) compromises long-term administration. Preclinical data suggest that the burden of critically short telomeres ( 21.9% CSTs) had 2-fold higher number of neuropathy ( P = 0.04) or fatigue ( P = 0.019) episodes and >3-fold higher number of myalgia episodes ( P = 0.005). The average telomere length was unrelated to the incidence of side effects. The percentage of CSTs, but not the average telomere size, is associated with weekly paclitaxel-derived toxicity.

Background: Rationalization of antiangiogenics requires biomarkers. Vascular re‐normalization is one widely accepted mechanism of action for this drug class. The interstitium of tumors with abnormal vasculature is hypoxic. We sought to track vascular normalization with 18F‐misonidazole ([18F]‐FMISO, a probe that detects hypoxia) PET, in response to window‐of‐opportunity (WoO) treatment with the antiangiogenic dovitinib. Methods: Two patient‐derived pancreas xenografts (PDXs; Panc215 and Panc286) and the spontaneous breast cancer model MMTV‐PyMT were used. Animals were treated during 1 week of WoO treatment with vehicle or dovitinib, preceded and followed by [18F]‐FMISO‐PET, [18F]‐FDG‐PET, and histologic assessment (dextran extravasation, hypoxia and microvessel staining, and necrosis, cleaved caspase‐3 and Ki67 measurements). After WoO treatment, gemcitabine (pancreas)/adriamycin (breast) or vehicle was added and animals were treated until the humane endpoint. Tumor growth inhibition (TGI) and survival were the parameters studied. Results: [18F]‐FMISO SUV did not change after dovitinib‐WoO treatment compared to vehicle‐WoO (0.54 vs. 0.6) treatment in Panc215, but it decreased significantly in Panc286 (0.58 vs. 1.18; P, Highlights We describe a new technique to monitor the efficacy of antiangiogenics.This drug class currently lacks biomarkers.Antiangiogenics exert their positive effect by inducing stromal normalization.18F‐fluoromisonidazole PET is able to monitor stromal normalization.Tumors showing stromal normalization by PET experience benefit from antiangiogenics.

2552Background: Our preclinical data show that one mechanism of acquired resistance to anti-angiogenic therapy involves hypoxia correction, measured by decreased SUV ( ↓ SUV) on FDG-PET followed by...

Objectives. To examine the prevalence of Human papillomavirus and Chlamydia trachomatis DNA in cervical samples among women with normal and abnormal cervical cytology from La Plata, Argentina. Methods. Two hundred and seventy-nine women (200 with cervical neoplasia or ICC and 79 women with normal cytology) provided cervical samples for the detection of HPV and C. trachomatis DNA by PCR-based assays. Results. HPV DNA increased with the cervical lesion severity, ranging from 30% among women with normal cytology to 99–100% among women with HSIL or ICC. C. trachomatis DNA prevalence increased from low levels in women with normal cytology (11%) to 47% in those with HSIL, but was uncommon among ICC patients (20%). Among women with normal cytology, C. trachomatis prevalence was higher in HPV DNA positive (12.5%) than HPV DNA negative women (10.9%), but this difference was not significant. Conclusions. HPV prevalence in the general population is slightly higher than those reported for other developing countries. C. trachomatis DNA positivity was associated with a higher risk of both LSIL and HSIL lesions, but not with ICC.

The aim of the present work was to evaluate the c-myc gene amplification process in cervical samples and to analyze the relationship between the activation of this proto-oncogene and the cytologic and/or histologic status. Thirty-four normal cervical samples and 105 abnormal cervical tissue scrapes, previously used for PAP or histopathologic diagnosis, were analyzed for c-myc gene amplification. Detection of c-myc gene amplification was performed using a polymerase chain reaction (PCR)-based method known as target arbitrarily primed-PCR (TAP-PCR). For c-myc amplification, significant differences were found between normal samples and samples presenting different grades of lesions (P

The aim of the present study was to evaluate the relationship between viral type and copy number of human papillomavirus (HPV) with respect to the grade of cervical disease, and also to identify the existence of an HPV type-dependent viral load effect. DNA from 275 exocervical specimens, previously evaluated for histologic diagnosis, were evaluated for HPV presence, HPV type, and viral load. Viral load determination was performed using the low stringency PCR method (LS-PCR). Significant differences were found between the samples infected with HPV16 with respect to the samples infected with other 'high-risk' viral types (HPV -18, -31, -33 or -51) and 'low-risk' types (P < 0.05). However, highly significant differences were found between the viral loads observed in the high-grade squamous intraepithelial lesions group and normal epithelium (OR = 8.53) or the low grade ones (OR = 3.10). Moreover, a high viral load was detected in the condyloma acuminatum group compared to the normal epithelia samples (p< 0.05). This work confirms the genotype-specific association of viral load to the presence of HPV16. Also, a trend to higher viral loads could be seen in the more compromised cervical lesions. An unexpected level of viral particles appeared associated to the condylomas. This fact could be explained by a productive infection with high levels of viral replication.