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.

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.

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.

Mitochondrial Inhibition: a Treatment Strategy in Cancer?

PURPOSE OF REVIEW: Mitochondria have a major impact on virtually all processes linked to oncogenesis. Thus, mitochondrial metabolism inhibition has emerged as a promising anticancer strategy. In this review, we discuss the anticancer potential of mitochondrial inhibitors, with particular focus on metformin, in the context of more effective, targeted therapeutic modalities, and diagnostic strategies for cancer patients. RECENT FINDINGS: Metformin has gained interest as an antitumor agent. However, promising results have not been translated into remarkable advances in the clinical practice. Recent findings emphasize the need of providing a metabolic context in which mitochondrial inhibitors may elicit its anticancerous effects. In addition, mitochondria are critical regulators in orchestrating immune responses. Thus, the immunomodulatory effect of mitochondrial inhibitors should also be taken into account to optimize its clinical use. Targeting mitochondrial metabolic network represents a promising therapeutic strategy in cancer. However, there is a need to define the metabolic context in which mitochondrial inhibitors are more effective, as well as how the cross-talk between many immunological functions and mitochondrial functionality may be exploited for a therapeutic benefit in cancer patients.

FGFR1 amplification or overexpression and hormonal resistance in luminal breast cancer: rationale for a triple blockade of ER, CDK4/6, and FGFR1.

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 < 0.05). FGFR1 amplification or overexpression occurred regardless of the luminal subtype, but the incidence was higher in luminal B (16.3%) than A (6.6%) tumors; P < 0.05. The Kappa index for overexpression and amplification was 0.69 (P < 0.001). Twenty-four per cent of the patients showed either amplification and/or overexpression of FGFR1, what was associated to a hazard ratio for relapse of 2.6 (95% CI 1.44-4.62, P < 0.001). In vitro, hormonal deprivation led to FGFR1 overexpression. Primary FGFR1 amplification, engineered mRNA overexpression, or LTED-R-acquired FGFR1 overexpression led to resistance against hormonotherapy alone or in combination with the CDK4/6 inhibitor palbociclib. Blocking FGFR1 with the kinase-inhibitor rogaratinib led to suppression of Rb phosphorylation, abrogation of the cell cycle, and resistance-reversion in all FGFR1 models. CONCLUSIONS: FGFR1 amplification and overexpression are associated to similar adverse prognosis in hormone-positive breast cancer. Capturing all the patients with adverse prognosis-linked FGFR1 aberrations requires assessing both features. Hormonal deprivation leads to FGFR1 overexpression, and FGFR1 overexpression and/or amplification are associated with resistance to hormonal monotherapy or in combination with palbociclib. Both resistances are reverted with triple ER, CDK4/6, and FGFR1 blockade.

Emerging role of Fatty acid synthase in tumor initiation: implications for cancer prevention.

Targeting metabolic reprogramming has emerged as a promising strategy for therapeutic intervention in cancer. We identify that fatty acid synthase (FASN) is essential for cancer initiation playing a critical role in acquiring three-dimensional (3D) growth properties during transformation. In vivo inhibition of FASN before oncogenic activation prevents tumor development and invasive growth suggesting that FASN could be a potential target for cancer prevention.

Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells.

Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies.

In vivo phosphoproteomics reveals kinase activity profiles that predict treatment outcome in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, we use high-throughput phosphoproteomics to build a functional TNBC taxonomy. A cluster of 159 phosphosites is upregulated in relapsed cases of a training set (n = 34 patients), with 11 hyperactive kinases accounting for this phosphoprofile. A mass-spectrometry-to-immunohistochemistry translation step, assessing 2 independent validation sets, reveals 6 kinases with preserved independent prognostic value. The kinases split the validation set into two patterns: one without hyperactive kinases being associated with a >90% relapse-free rate, and the other one showing ≥1 hyperactive kinase and being associated with an up to 9.5-fold higher relapse risk. Each kinase pattern encompasses different mutational patterns, simplifying mutation-based taxonomy. Drug regimens designed based on these 6 kinases show promising antitumour activity in TNBC cell lines and patient-derived xenografts. In summary, the present study elucidates phosphosites and kinases implicated in TNBC and suggests a target-based clinical classification system for TNBC.

Personalising and targeting antiangiogenic resistance: a complex and multifactorial approach.

Pathological angiogenesis involves complex and dynamic interactions between tumour cells and other lineages existing in the microenvironment of the tumour. Preclinical and clinical data suggest that tumours can show dual, different adaptive responses against antiangiogenic agents: one successful adaptation is vascular normalisation, whereas the second adaptation is elicited through vascular trimming and increased hypoxia. These phenomena depend on the type of tumour and the type of agent. The classical approach for investigating acquired resistance against antiangiogenic agents is to identify compensatory signalling pathways emerging in response to VEGF blockade, which has led to the development of highly effective drugs; however, ultimately these drugs fail. Here we review how the dual stromal adaptive patterns determine the mechanisms of escape that go beyond the reprogramming of signal transduction pathways, which obliges us to investigate the tumour as an ecosystem and to develop uni- and multicompartmental models that explain drug resistance involving metabolic and immune reprogramming. We also propose a method for facilitating personalised therapeutic decisions, which uses 18F-fluoromisonidazole-positron emission tomography to monitor the dual stromal response in tumours of individual patients.

Targeting Tumor Mitochondrial Metabolism Overcomes Resistance to Antiangiogenics.

Epithelial malignancies are effectively treated by antiangiogenics; however, acquired resistance is a major problem in cancer therapeutics. Epithelial tumors commonly have mutations in the MAPK/Pi3K-AKT pathways, which leads to high-rate aerobic glycolysis. Here, we show how multikinase inhibitor antiangiogenics (TKIs) induce hypoxia correction in spontaneous breast and lung tumor models. When this happens, the tumors downregulate glycolysis and switch to long-term reliance on mitochondrial respiration. A transcriptomic, metabolomic, and phosphoproteomic study revealed that this metabolic switch is mediated by downregulation of HIF1α and AKT and upregulation of AMPK, allowing uptake and degradation of fatty acids and ketone bodies. The switch renders mitochondrial respiration necessary for tumor survival. Agents like phenformin or ME344 induce synergistic tumor control when combined with TKIs, leading to metabolic synthetic lethality. Our study uncovers mechanistic insights in the process of tumor resistance to TKIs and may have clinical applicability.

Selective activity over a constitutively active RET-variant of the oral multikinase inhibitor dovitinib: results of the CNIO-BR002 phase I-trial.

BACKGROUND: Given our preclinical data showing synergy between dovitinib and paclitaxel in preclinical models we conducted this phase I trial aiming to define the recommended phase II-dose (RP2D) on the basis of toxicity and pharmacodynamic criteria while searching for genetic variants that could sensitize patients to the regimen under study. PATIENTS AND METHODS: A 3+3 escalation schedule was adopted. Seriated FGF23 and dovitinib and paclitaxel pharmacokinetic profiles were determined along a single-agent dovitinib "priming-phase" followed by a dovitinib + paclitaxel combination phase. RECIST 1.1 criteria and NCI CTCAE V.4.0 were used. In fresh pre-treatment tumor biopsy samples, FGFR1, 2 and 3 amplifications were revealed by FISH probes; 32 missense variants were genotyped in tumors and peripheral blood mononuclear cells with Taqman genotyping assays (FGFR1-3 and RET). Constructs encoding for wild-type and variant genes associated with clinical benefit were transfected into HEK-293 cells for preclinical experiments checking constitutive activation and dovitinib sensitivity of the variants. RESULTS: twelve patients were recruited in three dose-levels. At level 1B (200 mg dovitinib 5-days-on/2-days-off plus 60 mg/m 2-week of paclitaxel) more than 50% FGF23 upregulation was observed and no dose-limiting-toxicities (DLTs) occurred. The most frequent toxicities were asthenia, neutropenia, nausea/vomiting and transaminitis. Two patients with progressive disease prior to trial inclusion achieved prolonged disease stabilization. Both had the germline variant G2071A in the RET gene, which led to constitutive activation of the protein product and Y-905 phosphorylation, both in transfectants and in patients with the alteration. This variant was sensitive to dovitinib; in addition both patients experienced progression upon medication withdrawal. CONCLUSIONS: Level 1B was the RP2D as it provided adequate pharmacodynamic exposure to dovitinib. The G2071A germline variant act as a genetic modifier that renders different tumors sensitive to dovitinib.

Combinatorial effects of microRNAs to suppress the Myc oncogenic pathway.

Many mammalian transcripts contain target sites for multiple miRNAs, although it is not clear to what extent miRNAs may coordinately regulate single genes. We have mapped the interactions between down-regulated miRNAs and overexpressed target protein-coding genes in murine and human lymphomas. Myc, one of the hallmark oncogenes in these lymphomas, stands out as the up-regulated gene with the highest number of genetic interactions with down-regulated miRNAs in mouse lymphomas. The regulation of Myc by several of these miRNAs is confirmed by cellular and reporter assays. The same approach identifies MYC and multiple Myc targets as a preferential target of down-regulated miRNAs in human Burkitt lymphoma, a pathology characterized by translocated MYC oncogenes. These results indicate that several miRNAs must be coordinately down-regulated to enhance critical oncogenes, such as Myc. Some of these Myc-targeting miRNAs are repressed by Myc, suggesting that these tumors are a consequence of the unbalanced activity of Myc versus miRNAs.

Multiple E2F-induced microRNAs prevent replicative stress in response to mitogenic signaling.

Transcription of microRNAs (miRNAs) is thought to be regulated similarly to that of protein-coding genes. However, how miRNAs are regulated during the cell division cycle is not well understood. We have analyzed the transcription profiles of miRNAs in response to mitogenic stimulation in primary fibroblasts. About 33% of the miRNAs expressed in these cells are induced upon exit from quiescence. Many of these miRNAs are specifically induced by E2F1 or E2F3 during the G(1)/S transition and are repressed in E2F1/3-knockout cells. At least four miRNA clusters, let-7a-d, let-7i, mir-15b-16-2, and mir-106b-25, are direct targets of E2F1 and E2F3 during G(1)/S and are repressed in E2F1/3-null cells. Interestingly, these miRNAs do not contribute to E2F-dependent entry into S phase but rather inhibit the G(1)/S transition by targeting multiple cell cycle regulators and E2F targets. In fact, E2F1 expression results in a significant increase in S-phase entry and DNA damage in the absence of these microRNAs. Thus, E2F-induced miRNAs contribute to limiting the cellular responses to E2F activation, thus preventing replicative stress. Given the known function of E2F of inducing other oncogenic miRNAs, control of miRNAs by E2F is likely to play multiple roles in cell proliferation and in proliferative diseases such as cancer.

Environmental and human health risk assessment of organic micro-pollutants occurring in a Spanish marine fish farm.

In this work the risk posed to seawater organisms, predators and humans is assessed, as a consequence of exposure to 12 organic micro-pollutants, namely metronidazole, trimethoprim, erythromycin, simazine, flumequine, carbaryl, atrazine, diuron, terbutryn, irgarol, diphenyl sulphone (DPS) and 2-thiocyanomethylthiobenzothiazole (TCMTB). The risk assessment study is based on a 1-year monitoring study at a Spanish marine fish farm, involving passive sampling techniques. The results showed that the risk threshold for irgarol concerning seawater organisms is exceeded. On the other hand, the risk to predators and especially humans through consumption of fish is very low, due to the low bioconcentration potential of the substances assessed.

Genetic and epigenetic silencing of microRNA-203 enhances ABL1 and BCR-ABL1 oncogene expression.

The mammalian genome contains several hundred microRNAs that regulate gene expression through modulation of target mRNAs. Here, we report a fragile chromosomal region lost in specific hematopoietic malignancies. This 7 Mb region encodes about 12% of all genomic microRNAs, including miR-203. This microRNA is additionally hypermethylated in several hematopoietic tumors, including chronic myelogenous leukemias and some acute lymphoblastic leukemias. A putative miR-203 target, ABL1, is specifically activated in these hematopoietic malignancies in some cases as a BCR-ABL1 fusion protein (Philadelphia chromosome). Re-expression of miR-203 reduces ABL1 and BCR-ABL1 fusion protein levels and inhibits tumor cell proliferation in an ABL1-dependent manner. Thus, miR-203 functions as a tumor suppressor, and re-expression of this microRNA might have therapeutic benefits in specific hematopoietic malignancies.