Detection of activating estrogen receptor gene (ESR1) mutations in single circulating tumor cells

Purpose: Early detection is essential for treatment plans before onset of metastatic disease. Our purpose was to demonstrate feasibility to detect and monitor estrogen receptor 1 (ESR1) gene mutations at the single circulating tumor cell (CTC) level in metastatic breast cancer (MBC). Experimental Design: We used a CTC molecular characterization approach to investigate heterogeneity of 14 hotspot mutations in ESR1 and their correlation with endocrine resistance. Combining the CellSearch and DEPArray technologies allowed recovery of 71 single CTCs and 12 WBC from 3 ER-positive MBC patients. Forty CTCs and 12 WBC were subjected to whole genome amplification by MALBAC and Sanger sequencing. Results: Among 3 selected patients, 2 had an ESR1 mutation (Y537). One showed two different ESR1 variants in a single CTC and another showed loss of heterozygosity. All mutations were detected in matched cell-free DNA (cfDNA). Furthermore, one had 2 serial blood samples analyzed and showed changes in both cfDNA and CTCs with emergence of mutations in ESR1 (Y537S and T570I), which has not been reported previously. Conclusions: CTCs are easily accessible biomarkers to monitor and better personalize management of patients with previously demonstrated ER-MBC who are progressing on endocrine therapy. We showed that single CTC analysis can yield important information on clonal heterogeneity and can be a source of discovery of novel and potential driver mutations. Finally, we also validate a workflow for liquid biopsy that will facilitate early detection of ESR1 mutations, the emergence of endocrine resistance and the choice of further target therapy. ©2017 AACR

The dynamics of circulating tumour DNA (ctDNA) during treatment reflects tumour response in advanced melanoma patients

Despite the introduction of targeted (BRAFi/MEKi) and immune checkpoint inhibitors (ICIs) has significantly reduced the recurrence rate and improved the overall survival (OS) of patients with Stage III and IV melanoma, only a percentage will benefit of durable disease control. The aim of this study was to examine whether the levels of circulating tumour DNA (ctDNA) in plasma of advanced melanoma patients undergoing BRAFi/MEKi or ICIs vary according to the patients' survival outcomes (i.e. progression-free survival (PFS) and OS) and disease progression. Plasma samples of Stage III-IV melanoma patients were collected at baseline (treatment initiation) and thereafter every 3 months. Circulating BRAFV600E/K and NRASQ61R/K mutations were analysed through droplet digital PCR (ddPCR, Bio-Rad) in a total of 177 plasma samples from 48 melanoma patients (19 Stage III, 29 Stage IV). Baseline ctDNA concentration was significantly associated with OS (HR = 1.003, 95% CI = 1.000–1.006, p = 0.043) and PFS (HR = 1.004, 95% CI = 1.000–1.007, p = 0.029) independent of clinical-prognostic confounders. For each unit increase in the ∆ctDNA (concentration difference between the last follow-up and baseline) there was a 24% increased risk of disease progression, irrespective of treatment type and stage at diagnosis (OR = 1.24, 95% CI = 1.03–1.49, p = 0.020, AUC = 0.93). Patients with reduction of ctDNA level from baseline to the last follow-up had longer OS (HR = 0.14; 95% CI = 0.05–0.44, p = 0.001) and PFS (HR = 0.08; 95% CI = 0.03–0.27, p < 0.0001) compared to patients with increased ctDNA, including adjustment for confounding factors. Our findings suggest that variation of ctDNA over time during melanoma treatment reflects the clinical outcome and tumour response to therapy and might be helpful in clinical monitoring

The Pathogenic RET Val804Met Variant in Acromegaly: A New Clinical Phenotype?

Several genetic investigations were conducted to identify germline and somatic mutations in somatotropinomas, a subtype of pituitary tumors. To our knowledge, we report the first acromegaly patient carrying a RET pathogenic variant: c.2410G&gt;A (rs79658334), p.Val804Met. Alongside the fact that the patient’s father and daughter carried the same variant, we investigated the clinical significance of this variant in the context of somatotropinomas and other endocrine tumors, reviewing the RET mutations’ oncogenic mechanisms. The aim was to search for new targets to precisely manage and treat acromegaly. Our case describes a new phenotype associated with the RET pathogenic variant, represented by aggressive acromegaly, and suggests consideration for RET mutation screening if NGS for well-established PitNET-associated gene mutations renders negative

Functional effect of Saffron supplementation and risk genotypes in early age-related macular degeneration: a preliminary report.

Abstract BACKGROUND: To determine whether the functional effects of oral supplementation with Saffron, a natural compound that proved to be neuroprotective in early age-related macular degeneration, are influenced by complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) risk genotypes. METHODS: Thirty-three early AMD patients, screened for CFH (rs1061170) and ARMS2 (rs10490924) polymorphisms and receiving Saffron oral supplementation (20 mg/day) over an average period of treatment of 11 months (range, 6--12), were longitudinally evaluated by clinical examination and focal electroretinogram (fERG)-derived macular (18[degree sign]) flicker sensitivity estimate. fERG amplitude and macular sensitivity, the reciprocal value of the estimated fERG amplitude threshold, were the main outcome measures. RESULTS: After three months of supplementation, mean fERG amplitude and fERG sensitivity improved significantly when compared to baseline values (p &lt; 0.01). These changes were stable throughout the follow-up period. No significant differences in clinical and fERG improvements were observed across different CFH or ARMS2 genotypes. CONCLUSIONS: The present results indicate that the functional effect of Saffron supplementation in individual AMD patients is not related to the major risk genotypes of disease

Unlocking the potential of Molecular Tumor Boards: from cutting-edge data interpretation to innovative clinical pathways

The emerging era of precision medicine is characterized by an increasing availability of targeted anticancer therapies and by the parallel development of techniques to obtain more refined molecular data, whose interpretation may not always be straightforward. Molecular tumor boards gather various professional figures, in order to leverage the analysis of molecular data and provide prognostic and predictive insights for clinicians. In addition to healthcare development, they could also become a tool to promote knowledge and research spreading. A growing body of evidence on the application of molecular tumor boards to clinical practice is forming and positive signals are emerging, although a certain degree of heterogeneity exists. This work analyzes molecular tumor boards’ potential workflows, figures involved, data sources, sample matrices and eligible patients, as well as available evidence and learning examples. The emerging concept of multi-institutional, disease-specific molecular tumor boards is also considered by presenting two ongoing nationwide experiences

Unraveling the complexity of HRD assessment in ovarian cancer by combining genomic and functional approaches: translational analyses of MITO16-MaNGO-OV-2 trial

Background: Ovarian cancer (OvC) constitutes significant management challenges primarily due to its late-stage diagnosis and the development of resistance to chemotherapy. The standard treatment regimen typically includes carboplatin and paclitaxel, with the addition of poly (ADP-ribose) polymerase inhibitors for patients with high-grade serous ovarian cancer (HGSOC) harboring BRCA1/2 mutations. However, the variability in treatment responses suggests the need to investigate factors beyond BRCA1/2 mutations, such as DNA repair mechanisms and epigenetic alterations. Notably, homologous recombination repair deficiency (HRD) is observed in an additional 20% of HGSOC cases, indicating a broader spectrum of DNA repair defects. Existing commercial HRD assays have certain limitations, prompting a global effort to develop new genomic and functional tests through academic research. Materials and methods: This study investigates, in the 187 high-grade serous and endometrioid tumors from the MITO16/MaNGO-OV-2 trial, academic HRD genomic tests in conjunction with a RAD51 immunofluorescence assay to assess functional activation of HRD. Additionally, the study incorporates analysis of microRNA-506 (miR-506) expression as a putative epigenetic effector. Results: The RAD51 test identified HRD in 73% of the samples and genomic HRD testing in 57%, with HRD identified in 45% of samples by both tests. The significant discrepancy between the two assays emphasizes the need to refine tumor classification for HRD. A three-group genomic classification unveiled superior progression-free survival (PFS) in high- and mild-HRD tumors compared with negative-HRD tumors. High concordance between RAD51 and genomic testing in high-HRD tumors suggests a subset of 'super-HRD' tumors exhibiting superior PFS. High expression of miR-506 may be used to further refine HRD status. Conclusions: The study underscores the complexities of HRD assessment and advocates for a combined genomic and functional approach to enhance predictive accuracy in OvC treatment responses

Unraveling the complexity of HRD assessment in ovarian cancer by combining genomic and functional approaches: translational analyses of MITO16-MaNGO-OV-2 trial

Background: Ovarian cancer (OvC) constitutes significant management challenges primarily due to its late-stage diagnosis and the development of resistance to chemotherapy. The standard treatment regimen typically includes carboplatin and paclitaxel, with the addition of poly (ADP-ribose) polymerase inhibitors for patients with high-grade serous ovarian cancer (HGSOC) harboring BRCA1/2 mutations. However, the variability in treatment responses suggests the need to investigate factors beyond BRCA1/2 mutations, such as DNA repair mechanisms and epigenetic alterations. Notably, homologous recombination repair deficiency (HRD) is observed in an additional 20% of HGSOC cases, indicating a broader spectrum of DNA repair defects. Existing commercial HRD assays have certain limitations, prompting a global effort to develop new genomic and functional tests through academic research. Materials and methods: This study investigates, in the 187 high-grade serous and endometrioid tumors from the MITO16/MaNGO-OV-2 trial, academic HRD genomic tests in conjunction with a RAD51 immunofluorescence assay to assess functional activation of HRD. Additionally, the study incorporates analysis of microRNA-506 (miR-506) expression as a putative epigenetic effector. Results: The RAD51 test identified HRD in 73% of the samples and genomic HRD testing in 57%, with HRD identified in 45% of samples by both tests. The significant discrepancy between the two assays emphasizes the need to refine tumor classification for HRD. A three-group genomic classification unveiled superior progression-free survival (PFS) in high- and mild-HRD tumors compared with negative-HRD tumors. High concordance between RAD51 and genomic testing in high-HRD tumors suggests a subset of ‘super-HRD’ tumors exhibiting superior PFS. High expression of miR-506 may be used to further refine HRD status. Conclusions: The study underscores the complexities of HRD assessment and advocates for a combined genomic and functional approach to enhance predictive accuracy in OvC treatment responses

BRCA1/2 Molecular Assay for Ovarian Cancer Patients: A Survey through Italian Departments of Oncology and Molecular and Genomic Diagnostic Laboratories

In Italy, 5200 new ovarian cancers were diagnosed in 2018, highlighting an increasing need to test women for BRCA1/2. The number of labs offering this test is continuously increasing. The aim of this study was to show the results coming from the intersociety survey coordinated by four different Clinical and Laboratory Italian Scientific Societies (AIOM, SIAPEC-IAP, SIBIOC, and SIGU). A multidisciplinary team belonging to the four scientific societies drew up two different questionnaires: One was targeted toward all Italian Departments of Medical Oncology, and the second toward laboratories of clinical molecular biology. This survey was implemented from September 2017 to March 2018. Seventy-seven out of 305 (25%) Departments of Medical Oncology filled our survey form. Indeed, 59 molecular laboratories were invited. A total of 41 laboratories (70%) filled in the questionnaire. From 2014 to 2017, 16 new molecular laboratories were activated. A total of 12,559 tests were performed in the year 2016, with a mean of 339 tests and a median of 254 tests per laboratory, showing a glimpse of an extreme low number of tests performed per year by some laboratories. In terms of the type and number of professionals involved in the pre- and post-test counseling, results among the onco-genetic team were heterogeneous. Our data show that the number of laboratories providing BRCA1/2 germline assays is significantly increased with further implementation of the somatic test coming soon. The harmonization of the complete laboratory diagnostic path should be encouraged, particularly in order to reduce the gap between laboratories with high and low throughput

Reduction of serum IGF-I levels in patients affected with Monoclonal Gammopathies of undetermined significance or Multiple Myeloma. Comparison with bFGF, VEGF and K-ras gene mutation

<p>Abstract</p> <p>Background</p> <p>Serum levels of IGF-I in patients affected with multiple myeloma (MM) have been scarcely studied. The present study is aimed to explore this point comparing 55 healthy subjects, 71 monoclonal gammopaties of uncertain significance (MGUS) and 77 overt MM patients. In the same subjects, basic FGF and VEGF, have been detected. All three mediators were analyzed in function of K-<it>ras </it>mutation and melphalan response. Concerning IGF-I, two representative monitoring examples have also been added.</p> <p>Methods</p> <p>Cytokine determinations were performed by commercially available ELISA kits, while K12-<it>ras </it>mutation was investigated on genomic DNA isolated from bone marrow cell specimens by RFLP-PCR assay.</p> <p>Results</p> <p>Significant reductions of IGF-I levels were observed in MGUS and MM as compared with healthy controls. In addition, MM subjects showed significantly decreased serum IGF-I levels than MGUS. Conversely, increasing levels were observed for bFGF and VEGF, molecules significantly correlated. A multivariate analysis corrected for age and gender confirmed the significant difference only for IGF-I values (P = 0.01). K12-<it>ras </it>mutation was significantly associated with malignancy, response to therapy and with significantly increased serum bFGF levels.</p> <p>Conclusion</p> <p>IGF-I reduction in the transition: Controls→MGUS→MM and changes observed over time suggest that IGF-I should be furtherly studied in future clinical trials as a possible monitoring marker for MM.</p