Activating ESR1 mutations are a major mechanism of resistance to aromatase inhibitors in hormone receptor-positive, HER2-negative metastatic breast cancer (mBC). International guidelines, including those from ASCO, NCCN, and ESMO, recommend liquid biopsy as the preferred approach for ESR1 mutation testing at progression on endocrine therapy, with digital PCR (dPCR) and next-generation sequencing (NGS) as the preferred analytical platforms. Although elacestrant was approved by the U.S. Food and Drug Administration together with Guardant360® Dx as its companion diagnostic, European regulatory frameworks allow the use of validated in-house assays for ESR1 testing, which are increasingly being implemented across clinical laboratories. To support the clinical implementation of ESR1 testing and improve analytical standardization in routine practice, we performed a European multicentre analytical verification study using dPCR- and NGS-based liquid biopsy workflows. Six referral institutions participated in this study. All laboratories verified dPCR workflows and four also verified NGS-based assays using standardized reference materials containing clinically relevant ESR1 mutations. Limit of detection (LoD) and limit of blank (LoB) were determined in each laboratory according to locally validated workflows following CLSI-based verification procedures. Analytical sensitivity and specificity were assessed across platforms, focusing on the two most frequently tested ESR1 hotspot mutations, p.Y537S and p.D538G. Total DNA input ranged from 10 to 30 ng per reaction for dPCR assays, while NGS input followed platform-specific requirements. LoD values ranged from 0.01

High-grade tubo-ovarian carcinoma (HGOC) that exhibits homologous recombination deficiency (HRD) comprises almost half of these tumors and shows a greater response to platinum-based chemotherapies and a sensitivity to PARP inhibitors. Therefore, experts believe that tumor testing for HRD should be carried out at the time of primary diagnosis or disease recurrence, if not performed earlier. This study aimed to evaluate HRD in a large series of patients with HGOC via the centralized Myriad MyChoice CDx and the decentralized SOPHiA Genetics DDM HRD Solution assays in a real-world clinical practice in Spain. With the Myriad MyChoice CDx Plus assay, 502 (38%) of the 1322 tumor samples analyzed were categorized as HRD positive; overall, 198 (15%) cases were not informative. With the decentralized SOPHiA Genetics DDM HRD assay, 1876 (39%) of the 4777 tumor tissue samples analyzed were HRD positive; of the 4777 cases, 606 (13%) were non-informative. The HRD and mutational status results for all cases were reported to clinicians from participant laboratories in less than 21 working days using the SOPHiA Genetics DDM HRD assay. This study shows that a decentralized HRD test, such as the SOPHiA Genetics DDM HRD Solution assay, is a reliable and robust assay to provide therapeutic guidance for ovarian cancer patient management in clinical practice, with optimal performance in terms of an informative proportion of cases, positive HRD detection, and an adequate turnaround time.

The anti-HER2 monoclonal antibody trastuzumab and new derivative formulations are the standard treatment for HER2-positive breast cancer. However, after 1 to 5 years of treatment, some patients acquire resistance to therapy, leading to relapse. The microRNA-449 family members were downregulated in HER2-positive breast cancer cell lines and low levels were associated with patients’ worse prognosis. Moreover, trastuzumab-resistant HER2-positive breast cancer cell lines showed lower microRNAs-449 and higher Fatty Acid Synthase (FASN) expression, compared to sensitive cell lines. The direct regulation of FASN by microRNA-449a and microRNA-449b-5p was demonstrated. Moreover, microRNAs-449 overexpression and FASN inhibition decreased cell proliferation and sensitized cells to trastuzumab treatment by inhibiting the PI3K/AKT signaling pathway. Together, these results suggest the microRNAs-449/FASN axis as a potential therapeutic target in combination with anti-HER2 agents to overcome trastuzumab resistance and to improve treatment response in HER2-positive breast cancer patients.

INTRODUCTION:EGFR-mutated non-small cell lung cancer (EGFRmut NSCLC) represents a heterogeneous group of tumors with varying clinical outcomes. Resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI), is inevitable, with emerging evidence suggesting that concurrent genomic alterations influence treatment efficacy. MATERIALS AND METHODS:This retrospective study analyzed 58 stage IV EGFRmut NSCLC patients treated with osimertinib across four hospitals in Madrid, Spain, between March 2021 and February 2023. Comprehensive genomic profiling was conducted using next-generation sequencing (NGS) to evaluate co-mutations. Kaplan-Meier survival curves and Cox regression were applied to assess progression-free survival (PFS) and overall survival (OS). RESULTS:A second co-mutation was identified in 44.1 % of patients, with TP53 (70 %) being the most frequent, followed by EGFR (11.5 %), PI3K (11.5 %), and MET amplifications (7.7 %). Patients with co-mutations exhibited significantly worse PFS compared to those with only EGFR mutations (HR: 8.0, 95 % CI: 1.81-35.4; p = 0.001). Specifically, TP53 co-mutations were associated with reduced PFS (HR: 21.6, 95 % CI: 2.77-169; p < 0.001) and a non-statistically significant trend toward worse OS (HR: 3.10, 95 % CI: 0.89-10.8; p = 0.062). DISCUSSION:This study highlights the prognostic impact of co-mutations, particularly TP53, in EGFRmut NSCLC treated with osimertinib. These findings underscore the need for novel therapeutic approaches and personalized treatment strategies, especially in subgroups with poor prognoses. Trials such as MARIPOSA and FLAURA-2 provide promising evidence for treatment intensification, but careful patient stratification is essential to balance efficacy and toxicity.

Abstract Objective This study aimed to describe the performance of a next‐generation sequencing (NGS) panel for the detection of precise genomic alterations in cancer in Spanish clinical practice. The impact of tumor characteristics was evaluated on informative NGS and actionable mutation rates. Materials and Methods A cross‐sectional study was conducted at the Fundación Jiménez Díaz University Hospital (May 2021–March 2022) where molecular diagnostic of 537 Formalin‐Fixed Paraffin‐Embedded (FFPE) tissue samples of diverse solid tumors (lung, colorectal, melanoma, gastrointestinal stromal, among others) was performed using AVENIO Tumor Tissue Targeted Kit. A descriptive analysis of the features of all samples was carried out. Multivariable logistic analysis was conducted to assess the impact of sample characteristics on NGS performance defined by informative results rate (for all tumors and for lung tumors), and on actionable mutations rate (for lung tumors only). Results AVENIO performance rate was 75.2% in all tumor samples and 75.3% in lung cancer samples, and the multivariable analysis showed that surgical specimens are most likely to provide informative results than diagnostic biopsies. Regarding the mutational findings, 727 pathogenic, likely pathogenic, or variant of unknown significance mutations were found in all tumor samples. Single nucleotide variant was the most common genomic alteration, both for all tumor samples (85.3% and 81.9% for all solid tumors and lung samples, respectively). In lung tumors, multivariable analysis showed that it is more likely to find actionable mutations from non‐smokers and patients with adenocarcinoma, large cell, or undifferentiated histologies. Conclusion This is the largest cohort‐level study in Spain to profile the analyses of biopsy samples of different tumors using NGS in routine clinical practice. Our findings showed that the use of NGS routinely provides good rates of informative results and can improve tumor characterization and identify a greater number of actionable mutations.

The impact of exercise on pediatric tumor biology is essentially unknown. We explored the effects of regular exercise on tumor proteome profile (as assessed with liquid chromatography with tandem mass spectrometry) in a mouse model of one of the most aggressive childhood malignancies, high-risk neuroblastoma (HR-NB). Tumor samples of 14 male mice (aged 6-8 wk) that were randomly allocated into an exercise (5-wk combined aerobic and resistance training) or nonexercise control group (6 and 8 mice/group, respectively) were analyzed. The Search Tool for the Retrieval of Interacting Genes/Proteins database was used to generate a protein-protein interaction (PPI) network and enrichment analyses. The Systems Biology Triangle (SBT) algorithm was applied for analyses at the functional category level. Tumors of exercised mice showed a higher and lower abundance of 101 and 150 proteins, respectively, than controls [false discovery rate (FDR) < 0.05]. These proteins were enriched in metabolic pathways, amino acid metabolism, regulation of hormone levels, and peroxisome proliferator-activated receptor signaling (FDR < 0.05). The SBT algorithm indicated that 184 and 126 categories showed a lower and higher abundance, respectively, in the tumors of exercised mice (FDR < 0.01). Categories with lower abundance were involved in energy production, whereas those with higher abundance were related to transcription/translation, apoptosis, and tumor suppression. Regular exercise altered the abundance of hundreds of intratumoral proteins and molecular pathways, particularly those involved in energy metabolism, apoptosis, and tumor suppression. These findings provide preliminary evidence of the molecular mechanisms underlying the potential effects of exercise in HR-NB. NEW & NOTEWORTHY We used liquid chromatography with tandem mass spectrometry to explore the impact of a 5-wk exercise intervention on the tumor proteome profile in a mouse model of one of the most aggressive childhood malignancies, high-risk neuroblastoma. Exercise altered the abundance of hundreds of proteins and pathways, particularly those involved in energy metabolism and tumor suppression. These molecular changes could mediate, at least partly, the potential antitumorigenic effects of exercise.

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PDF file - 119K, Supplementary Table 1. Patients characteristics. Supplementary Table 2. Biomarker expression in human samples. Supplementary Table 3. Spearman correlation between biomarkers in cases showing expression. Supplementary Table 4. Association of Met and p-Met with other biomarkers. Supplementary Table 5. Cox regression analysis for overall survival for each variable adjusted for performance status (PS) and stage. Supplementary Table 6. Biomarker status in biopsies pre-treatment and at relapsed disease.

Supplementary Figure S1. MKP-1 protein abundance levels in MDA-MB-231 cells treated with docetaxel at progressively increasing times. Supplementary Figure S2. Cell growth in the presence of the docetaxel or doxorubicin of breast cancer cell lines overexpressing MKP-1. Supplementary Table S1. Clinical and molecular characteristics of a series of 350 patients with early breast cancer. Supplementary Figure S3. IHC detection of MKP-1 and p-JNK1/2 in a representative normal tissue sample. Supplementary Table S2. Univariate and multivariate Cox analyses in the cohort of 350 breast cancer patients (disease-free survival analysis). Supplementary Table S3. Clinical and molecular characteristics of 64 locally advanced breast cancer patients who received neoadjuvant taxane-based chemotherapy.Supplementary Table S4. MKP-1 and p-JNK1/2 marker determinations in locally advanced breast cancer patients who received neoadjuvant taxane-based chemotherapy.

XLS - 262K, Differentially expressed transcripts between true interval cancers and screen-detected cancers at p-value<0.05.

Representative immunohistochemistry images of control and 765 T-DM1 treated explant obtained from a liver metastasis.

Homologous Recombination Deficiency (HRD) is assessed by detection of mutations in BRCA1/2 genes (BRCAm) and/or genomic instability score (GIS+). In ovarian cancer, HRD has become an important biomarker to stratify patients for appropriate clinical management and treatment. Myriad myChoice CDX Plus is currently the gold standard for assessing HRD on tumor samples. There is an increasing interest in transitioning into inhouse HRD assays that fulfill analytical concordance and, also, demonstrate clinical validation. Sophia DDM Dx HRD solution is one of the alternatives, which has reached an overall percent agreement of 93% with Myriad, demonstrating also clinical validation in PAOLA-1 trial. In this study, we present the real-world results of HRD testing in Spain. Phase 1 (Apr2021-Jun22), consisted in the analysis of 1322 cases from 90 centers, using Myriad myChoice. Phase 2 (Jul2022-Dec22) included 1333 cases from 135 centers, analyzed with Sophia HRD solution in 4 academic centers, after an internal validation. Phase 1 reported 47% BRCAwt/GIS-, 24% BRCAwt/GIS+, 14% BRCAm (BRCAm/GIS- y BRCAm/GIS+), and 15% inconclusive cases using myChoice. In phase 2, Sophia HRD identified 47% BRCAwt GIS-, 25% BRCAwt/GIS+, 15% BRCAm, and 13% inconclusive cases. The distribution of HRP (range 42.4-50%) and HRD (range 35.7- 40.7%) cases was similar among the reference centers. In addition to GIS and BRCA1/2, Sophia reported CCNE1 amplification (5.3%), as well as mutations in BRIP1 (2.4%), RAD51C (1.1%), RAD51D (1.2%) and PALB2 (1.9%). As expected, CCNE1 amplification was more frequent in HRP (86.5%) than HRD (13.5%). The results obtained from a cohort of 2655 ovarian cancer samples by two different methods, Myriad myChoice and Sophia HRD, confirm the analytical concordance between HRD assays. Percentages of BRCAwt/GIS-, BRCAwt/GIS+, BRCAm, and invalid cases were almost identical. In conclusion, Sophia DDMTM Dx HRD solution shows a high interlaboratory agreement, providing clinically relevant molecular information, and improving the interaction between referral and reference centers.

PDF - 2988K, Supplementary Figure 1. Levels of immunoprecipitated PP2A from lysates of 21 CRC patients used in PP2A phosphatase assays (1D6 antibody). Supplementary Figure 2. Quantification of PP2A activity in CRC patients with normal mucosa and tumor samples (FL-309 antibody); * P < 0.05; ** P < 0.01; N: normal mucosa; T: tumor sample. Supplementary Figure 3. Levels of immunoprecipitated PP2A from lysates of 21 CRC patients used in PP2A phosphatase assays (FL-309 antibody). Supplementary Figure 4. Comparison between normal PPP2R1B sequence and silent alterations at codons 72 (A) and 391 (B) found in two CRC patients. The position altered is indicated by an arrow. Supplementary Figure 5. Western blot analysis of PPP2R2A and PPP2R5E in 21 CRC patients. Supplementary Figure 6. Quantification of PP2A activity using the 1D6 and FL-309 antibodies and western blot analysis of PP2A, CIP2A and SET in 7 CRC cell lines; * P < 0.05; ** P < 0.01; N1-2, normal tissue. Supplementary Figure 7. FTY720 treatment induces a PP2A activation. (A) Treatment with OA inhibits the FTY720-induced PP2A activity in RKO and LoVo cells. (B) Levels of immunoprecipitated PP2A (FL-309 antibody) from the RKO and LoVo lysates used in the phosphatase assays. Supplementary Figure 8. Analysis of cell viability in SW480, HT-29 and DLD-1 cells after FTY720 treatment. Supplementary Figure 9. Optical microscope images showing changes in morphology and cell viability after FTY720 treatment in RKO and DLD-1 cells. Supplementary Figure 10. Colony forming ability of HT-29 and DLD-1 cells untreated or FTY720-treated alone or in combination with okadaic acid. Supplementary Figure 11. Western blot showing PP2A, AKT and ERK1/2 phosphorylation and expression after FTY720 treatment in HT-29 and SW480 cells. Supplementary Figure 12. Western blot analysis of CIP2A, c-MYC, SET and PP2A expression after FTY720 treatment in RKO and LoVo cells. Supplementary Figure 13. MTS assays showing the effect of FTY720 and 5-FU (A), SN-38 (B) or LOHP (C) alone or in combination in DLD-1 cells. Supplementary Figure 14. MTS assays showing the effect of FTY720 and 5-FU (A), SN-38 (B) or LOHP (C) alone or in combination in HT-29 cells. Supplementary Figure 15. Effects of FTY720 on proliferation and apoptosis in a total of 5 sets of human samples, including normal mucosa and colorectal tumor. Each row represents an effect on expression of the paired control and FTY720 treated samples, and each column, a single IHC marker, including Ki-67 and cleaved caspase 3. Downregulation of expression is displayed in green, upregulation in red, and no effect in black.

Trastuzumab treatment has significantly improved the prognosis of HER2-positive breast cancer patients. Despite this, resistance to therapy still remains the main clinical challenge. In order to evaluate the implication of microRNAs in the trastuzumab response, we performed a microRNA array in parental and acquired trastuzumab-resistant HER2-positive breast cancer cell lines. Our results identified miR-146a-5p as the main dysregulated microRNA. Interestingly, high miR-146a-5p expression in primary tumor tissue significantly correlated with shorter disease-free survival in HER2-positive breast cancer patients. The gain- and loss-of-function of miR-146a-5p modulated the response to trastuzumab. Furthermore, the overexpression of miR-146a-5p increased migration and angiogenesis, and promoted cell cycle progression by reducing CDKN1A expression. Exosomes from trastuzumab-resistant cells showed a high level of miR-146a-5p expression compared with the parental cells. In addition, the co-culture with resistant cells’ exosomes was able to decrease in sensitivity and increase the migration capacities in trastuzumab-sensitive cells, as well as angiogenesis in HUVEC-2 cells. Collectively, these data support the role of miR-146a-5p in resistance to trastuzumab, and demonstrate that it can be transferred by exosomes conferring resistance properties to other cells.

PDF file - 354K, PF-2341066 sensitizes H841-derived xenografts cells to chemotherapy.

PDF file - 441K, Cellular and molecular changes induced by Met inhibitors.

Supplementary Table S1. Clinical and molecular characteristics of a series of 242 patients with metastatic CRC.

PDF file - 40K, Kaplan-Meier curves for overall survival univariate analysis according to biomarker status.

Supplementary Figure S4. Effects of FTY720 in SW480, HT-29 and LS513 cells ectopically expressing SET and treated with 5-FU alone or combined with oxaliplatin.

PDF file - 126K, PF-2341066 treatment upfront decreases tumorogenesis and local invasion in H69M xenograft.