High mechanical conditioning by tumor extracellular matrix stiffness is a predictive biomarker for anti-fibrotic therapy in HER2-negative breast cancer.

BACKGROUND: Tumor progression has been linked to stiffening of the extracellular matrix (ECM) caused by fibrosis. Cancer cells can be mechanically conditioned by stiff ECM, exhibiting a 1004-gene signature (MeCo score). Nintedanib has demonstrated anti-fibrotic activity in idiopathic pulmonary fibrosis. This study explores nintedanib's anti-fibrotic effect on breast cancer outcomes. METHODS: We present long-term follow-up and analysis of a neoadjuvant randomized Phase 2 trial in early HER2-negative breast cancer. Patients (N = 130) underwent a baseline biopsy and received 12 paclitaxel courses alone (control arm) or in combination with nintedanib (experimental arm). Tumor MeCo score was determined by RNAseq. The primary aim was to assess nintedanib's impact on event-free survival (EFS) based on MeCo scores. RESULTS: Follow-up data were retrieved from 111 patients; 75 baseline and 24 post-run-in phase samples were sequenced. After median follow-up of 9.67 years, median EFS was not statistically different between arms (P = 0.37). However, in the control arm, High versus Low MeCo patients had a statistically higher relapse risk: hazard ratio (HR) = 0.21; P = 0.0075. This risk was corrected by nintedanib in the experimental arm: HR = 0.37; P = 0.16. Nintedanib demonstrated pharmacodynamic engagement, reducing the MeCo score by 25% during the run-in phase (P<0.01). Patients with Low MeCo after run-in had the best long-term prognosis (HR = 0.087; P = 0.03). CONCLUSIONS: High MeCo is predictive of poor outcomes in HER2-negative early breast cancer, although this risk can be mitigated by nintedanib, which is able to specifically reduce mechanical conditioning.

Distribution of PD-L1, TROP2 and HER2- "lowness" in early triple-negative breast cancer: an opportunity for treatment de-escalation.

BACKGROUND: HER2, TROP2 and PD-L1 are novel targets in triple-negative breast cancer (TNBC). The combined expression status of these targets, and whether they can define prognostic subgroups, is currently undefined. METHODS: Immunohistochemistry was used to determine HER2, TROP2 and PD-L1 levels in 459 TNBC cases, that received in the adjuvant/neoadjuvant setting active surveillance, CMF, anthracycline-, anthracycline plus taxane-, or carboplatin-containing regimes. RESULTS: HER2-low patients with PD-L1 > 1 CPS (double-positive, herein "DP") had a mean PFS of 4768 days (95% CI: 4267-5268) versus 3522 days (95% CI: 3184-3861) for non-DP patients (P = 0.002). Regarding the received adjuvant treatment, DP patients (versus non-DP) receiving anthracyclines plus taxanes exhibited a mean PFS time of 4726 (95% CI: 4022-5430) versus 3302 (95% CI: 2818-3785) days (P = 0.039). Finally, 100% of DP patients that received a carboplatin-based regimen were long-term disease-free. CONCLUSIONS: Early HER2-low, PD-L1-positive TNBC patients have a very good prognosis, particularly if treated with anthracycline/taxane- or carboplatin-containing regimes.

Usefulness and real-world outcomes of next generation sequencing testing in patients with cancer: an observational study on the impact of selection based on clinical judgement.

Background: Next Generation Sequencing (NGS) panels are increasingly used in advanced patients with cancer to guide therapy. There is, however, controversy about when should these panels be used, and about their impact on the clinical course. Methods: In an observational study of 139 patients with cancer having an NGS test [from January 1st, 2017 to December 30th, 2020, in two hospitals (Hospital Universitario de La Princesa and Hospital Universitario Quironsalud Madrid) from Spain], we evaluated whether the clinical course (progression-free survival, PFS) was influenced by drug-based criteria [druggable alterations, receiving a recommended drug, having a favourable ESCAT category (ESMO Scale for Clinical Actionability of molecular Targets)] or clinical judgement criteria. Findings: In 111 of 139 cases that were successfully profiled, PFS was not significantly influenced by either having druggable alterations [median PFS for patients with druggable alterations was 170 (95% C.I.: 139-200) days compared to 299 (95% C.I.: 114-483) for those without; p = 0.37], receiving a proposed matching agent [median PFS for patients receiving a genomics-informed drug was 195 days (95% C.I.: 144-245), compared with 156 days for those that did not (95% C.I.: 85-226); p = 0.50], or having favourable ESCAT categories [median PFS for patients with ESCAT I-III was 183 days (95% C.I.: 104-261), compared with 180 (95% C.I.:144-215) for patients with ESCAT IV-X; p = 0.87]. In contrast, NGS testing performed within clinical judgement showed a significantly improved PFS [median PFS for patients that were profiled under the recommended scenarios was 319 days (95% C.I.: 0-658), compared to 123 days (95% C.I.: 89-156) in the non-recommended categories; p = 0.0020]. Interpretation: According to our data, real-world outcomes after NGS testing provide evidence of the benefit of clinical judgement in patients with either advanced cancers that routinely need multiple genetic markers, patients with advanced rare cancers, or patients that are screened for molecular clinical trials. By contrast, NGS does not seem to be valuable when performed in cases with a poor PS, rapidly progressing cancer, short expected lifetime, or cases with no standard therapeutic options. Funding: RC, NR-L and MQF are recipients of the PMP22/00032 grant, funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF). The study also received funds from the CRIS Contra el Cancer Foundation.

RANK is a poor prognosis marker and a therapeutic target in ER-negative postmenopausal breast cancer.

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor, denosumab, in breast cancer patients, beyond the bone, is unclear. Aiming to select patients who may benefit from denosumab, we hereby analyzed RANK and RANKL protein expression in more than 2,000 breast tumors (777 estrogen receptor-negative, ER- ) from four independent cohorts. RANK protein expression was more frequent in ER- tumors, where it associated with poor outcome and poor response to chemotherapy. In ER- breast cancer patient-derived orthoxenografts (PDXs), RANKL inhibition reduced tumor cell proliferation and stemness, regulated tumor immunity and metabolism, and improved response to chemotherapy. Intriguingly, tumor RANK protein expression associated with poor prognosis in postmenopausal breast cancer patients, activation of NFKB signaling, and modulation of immune and metabolic pathways, suggesting that RANK signaling increases after menopause. Our results demonstrate that RANK protein expression is an independent biomarker of poor prognosis in postmenopausal and ER- breast cancer patients and support the therapeutic benefit of RANK pathway inhibitors, such as denosumab, in breast cancer patients with RANK+ ER- tumors after menopause.

p27Kip1 V109G as a biomarker for CDK4/6 inhibitors indication in hormone receptor-positive breast cancer.

CDK4/6 inhibitors benefit a minority of patients who receive them in the breast cancer adjuvant setting. p27Kip1 is a protein that inhibits CDK/Cyclin complexes. We hypothesized that single-nucleotide polymorphisms that impaired p27Kip1 function could render patients refractory to endocrine therapy but responsive to CDK4/6 inhibitors, narrowing the patient subpopulation that requires CDK4/6 inhibitors. We found that the p27Kip1 V109G single-nucleotide polymorphism is homozygous in approximately 15% of hormone-positive breast cancer patients. Polymorphic patients experience rapid failure in response to endocrine monotherapy compared with wild-type or heterozygous patients in the first-line metastatic setting (progression-free survival: 92 vs 485 days, P < .001); when CDK4/6 inhibitors are added, the differences disappear (progression-free survival: 658 vs 761 days, P = .92). As opposed to wild-type p27Kip1, p27Kip1 V109G is unable to suppress the kinase activity of CDK4 in the presence of endocrine inhibitors; however, palbociclib blocks CDK4 kinase activity regardless of the p27Kip1 status. p27Kip1 genotyping could constitute a tool for treatment selection.

Tumor P70S6K hyperactivation is inversely associated with tumor-infiltrating lymphocytes in triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is characterized by large heterogeneity and relative lack of available targeted therapies. To find therapeutic strategies for distinct patients with TNBC, several approaches have been used for TNBC clustering, including recently immune and phosphoproteomic patterns. Based on 70-kDa ribosomal protein S6 kinase (P70S6K)-TNBC clustering, the current study explores the immune profiling in TNBC tumors. METHODS: Stromal tumor-infiltrating lymphocytes (sTILs) were evaluated in human TNBC tumor samples. Furthermore, immunohistochemistry staining for CD8, CD4, Foxp3, and CD20 was performed in tissue microarrays (TMA) sections. RESULTS: Histological analysis showed decreased sTILs, CD20+ cells, and CD8+/CD4+ ratio in high phosphorylated P70S6K (p-P70S6K) tumors. Moreover, p-P70S6K score was directly correlated with CD4+ and Foxp3+ T cells, while it was inversely correlated with CD8+/CD4+ and CD8+/Foxp3+ ratios. CONCLUSION: sTIL infiltration and lymphocyte profiling vary in the context of hyperactivation of P70S6K in TNBC tumors.

A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis.

We report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere.

Prolonged estrogen deprivation triggers a broad immunosuppressive phenotype in breast cancer cells.

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.

[6]-Gingerol-Derived Semi-Synthetic Compound SSi6 Inhibits Tumor Growth and Metastatic Dissemination in Triple-Negative Breast Cancer Xenograft Models.

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.

Monitoring vascular normalization: new opportunities for mitochondrial inhibitors in breast cancer.

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.