TEST:

Oncomine™ Pan-Cancer Cell-Free Assay

Blood-based LB performed by NGS is a non-invasive viable alternative tool for molecular genotyping and identifiy tumor-derived somatic alterations to increase the number of pts elegible to target therapy and guide personalized medicine.

TRIPLETE is a phase III trial where 435 pts with RAS/BRAF wt – per local assessment – mCRC were randomized to receive first-line FOLFOX/ panitumumab (pan) or mFOLFOXIRI/pan... In contrast to previous data, negative hyperselection both through tissue and plasma analysis failed to demonstrate a prognostic role among pMMR RAS/BRAF wt mCRC pts treated with first-line chemotherapy plus pan. The confounding effect of the associated chemotherapy is a potential explanation for this finding. Tissue and plasma analyses at baseline failed to provide fully concordant results.

The assay shows good performance in detecting hotspot SNVs and small indels in ctDNA, and is suitable for clinical use. The limitation of bioinformatics pipelines in variant calling should be noted, and consideration can be given to run multiple pipelines in parallel to avoid missing out variants.

MPS-based molecular testing shows promise in identifying actionable genomic alterations, potentially enabling the stratification of patients for targeted chemotherapeutic treatments. Future research should focus on integrating OCA and OPCCFA testing, as well as similar MPS-based assays, into existing surveillance and management protocols for patients with bile duct strictures.

By being complementary or when tissue is not available, liquid biopsy has great potential in lung cancer management, ultimately improving patient outcomes and treatment efficacy. Type of detected mutations Mutations Targetable n(%) Non-targetable n(%) EGFR 8 (47.1) KRAS 6 (35.2) 4 (26.6) BRAF 1 (5.9) 1 (6.7) ERBB2 1 (5.9) MET ex 14 1 (5.9) FGFR4 1 (6.7) GNAS 2 (13.3) RET 1 (6.7) MAP2K1 1 (6.7) TP53 5 (33.3)

The integration of baseline and early on-treatment ctDNA analysis enables the identification of patients that do not benefit from IO-based treatments as early as three weeks after the initiation of the therapy, anticipating disease progression and being useful to tailor patient management.

A high proportion of IBC patients had persistent MRD despite achieving pCR. Longitudinal assessment of the molecular landscape is necessary for optimal risk stratification in patients with IBC.

TRIPLETE is a phase III trial where 435 pts with RAS/BRAF wt mCRC were randomized to receive first-line FOLFOX/panitumumab (pan) or mFOLFOXIRI/pan. Pts enrolled in the TRIPLETE trial with available tissue and plasma at baseline (BP), and plasma at the time of disease progression (PDP) were included... In contrast to previous data, negative hyperselection both through tissue and plasma analysis failed to demonstrate a prognostic role among pMMR RAS/BRAF wt mCRC pts treated with first-line chemotherapy plus pan. The confounding effect of the associated chemotherapy is a potential explanation for this finding.

Molecular analysis of CTCs is a challenge since CTCs are very rare in the blood and there are technical limitations in isolating CTCs with sufficient purity for NGS analysis. In this study, we developed the process for NGS analysis on very small number of high-purity CTCs using CytoGen's Smart Biopsyâ„¢ System. Taken together with the results from our ongoing clinical trials, this process could provide information for the diagnosis and appropriate treatment of breast cancer and NSCLC patients.

TRIPLETE is a phase III trial where 435 pts with untreated RAS/BRAF wt – per local assessment - mCRC were randomized to receive first-line FOLFOX/panitumumab (pan) or mFOLFOXIRI/pan... Inconsistently with previous data, negative hyperselection both through tissue and plasma analysis failed to demonstrate a prognostic role among pMMR RAS/BRAF wt mCRC pts treated with first-line chemotherapy plus pan. The confounding effect of the associated chemotherapy backbone is a potential explanation for this finding. FFPE tissue and plasma analyses at baseline failed to provide fully concordant results.

Molecular analysis of CTCs is a challenge since CTCs are very rare in the blood and there are technical limitations in isolating CTCs with sufficient purity. In this study, we developed the process for NGS analysis on very low number of CTCs with high purity, isolated using CytoGen's Smart Biopsyâ„¢ system. Overall, these process could provide information for diagnosis and appropriate treatment of metastatic breast cancer along with the results of our ongoing clinical trials.

Conclusion This study shows the feasibility of using a gene panel to identify ctDNA positive patients which will facilitate its use on a larger scale. ctDNA positivity was significantly associated with worse oncological outcome.

The study of longitudinal cfDNA samples with the NGS panel in EC allowed us to understand how the mutational landscape of patients behaves throughout time, with the arisen of novel mutations not found on the baseline samples, that may derive from treatment pressure. Conclusion The study of longitudinal cfDNA samples with the NGS cell-free panel represents a comprehensive strategy for a non-tumour informed characterization of endometrial tumours that reflects the clinical evolution and even allows for the identification of novel mutations that could be used as actionable targets.

Evidence suggests that cfDNA analysis can provide accurate and reliable information on somatic genomic alterations in patients with GC, potentially replacing tissue biopsy as a diagnostic and prognostic tool. Through cfDNA analysis for molecular profiling, it may be possible to translate the molecular classification into therapeutic targets and predictive biomarkers, leading to personalized treatment options for patients with GC in the future.

Bile mutations are the same found in tissue samples of CCAs (KRAS, TP53, ERBB3, GNAS, FBXW7, ERB2, IDH2, MAPK2K1 and FGFR3). In clinical suspected malignant strictures, diagnosed as benign or indeterminate, assessment for bile mutations could determine the clinical follow up and is a strong indicator for repeating cytology or biopsy in order to avoid a false negative diagnosis.

ER/PGR/Ki67 percentage; cerbB2 ASCO/CAP/2018score. Tissue biopsies(24) and plasma(8) served for DNA/RNA extraction: RecoverAll/Total/Nucleic/Acid/Isolation/kit and Thermo-Fisher, MagMAXCell-free/Total/Nucleic/Acid/Kit. DNA/RNA concentration: Invitrogen, Qubit/RNA/HS/Assay/kit, Qubit-1X-ds-DNA-HS-Assay and Qubit-4-Fluorometer.

This is an initial experience from India using liquid biopsy NGS for dynamic monitoring in immunotherapy treated patients. The ctDNA concentration was predictive of response as well as impending progression. A 50% cut-off for response as well as a baseline ctDNA 0f 0.3ng/microlitre showed a trend for better PFS in these patients.

Introduction: Treatment with neoadjuvant nivolumab plus chemotherapy has demonstrated high efficacy in patients with locally advanced non-small-cell lung cancer (NSCLC)... Our data suggest that BRAF pathogenic variants may be a good prognostic factor in locally advance NSCLC patients treated with neoadjuvant chemo-immunotherapy.

Molecular profiling of liquid biopsies at baseline identified alterations that could impact on patient outcomes. Our data suggest that the presence of BRAF pathogenic variants may be a good prognostic factor in stage IV NSCLC patients treated with immunotherapy or chemo-immunotherapy, while pathogenic mutations in ERBB3 could be a biomarker of high risk of disease progression. Additional studies analyzing larger cohorts of patients are needed to clarify these hypotheses.

Quantification of ctDNA levels for prognostic estimation should be adapted for each methodology.

CfDNA from pleural fluid supernatant is adequate to identify tumor molecular markers found in cell block and can possibly even act as a supplement to increase detection of therapy altering mutations. With further study, this new tool for evaluating MPEs may help in reducing future invasive procedures.

Osimertinib was used in 59% (16/27), Dacomitinib in 4% (1/27) and Gefitinib/Erlotinib in 37% (10/27). Clearance of pEGFR was predictive of improved TTTF. Our results support using NGS at diagnosis and ddPCR for monitoring response, whereas decrease in CEA levels could be explored as a surrogate for pEGFR clearance.

Conclusions The use of liquid biopsy in early stages and the follow-up of patients with NSCLC is a potential tool for detecting tumor recurrence, as demonstrated by the increase in cfDNA concentration, mutated allele frequency and gene dosage after relapse. A long-term follow-up is required to assess the consistency and robustness of results.

"Conclusion Our preliminary results show good correlation of gene mutation and HPV detection between tumor tissue and baseline LB at initial diagnosis. We will continue patient recruitment and start the analysis of sequential LB after tumor treatment and during follow-up."

Similarly, mutations in NRAS and HRAS were more frequently detected in samples from tumors harboring mutations in exons 18 or 21 (Fisher p-value: 0.050 and Fisher p-value: 0.099, respectively). In conclusion, our data suggest that the mechanisms underlying EGFR-TKI resistance could be dependent on the exon location of the original EGFR-sensitizing mutation.

The present study demonstrates that cfDNA and TP53 are potential predictive biomarkers of response in mccRCC to be further explored in larger and/or prospective studies.

Table: 589P Baseline clinicopathological data (* four patients excluded due to suspected germline mutation) Conclusions In this series of ctDNA from EC patients, 29% of the patients were considered ctDNA positive. ctDNA positivity was prognostic for risk of recurrence and warrant longitudinal analysis in EC.

"We tested plasma samples from the Erbitux Metastatic Colorectal Cancer Strategy (ERMES) phase III trial of FOLFIRI+Cetuximab in first-line treatment of RAS/BRAF wild-type mCRC...These data confirm that the occurrence of RAS/BRAF mutations in mCRC patients receiving anti-EGFR agents is relatively frequent. However, the cfDNA dynamics of RAS mutations in patients treated with anti-EGFR agents plus polychemotherapy are complex and might not be directly associated with resistance to treatment."

Methods Using samples from NADIM clinical trial (NCT03081689), in which resectable stage IIIA NSCLC patients were treated with neoadjuvant chemo-immunotherapy with nivolumab, we have evaluated the capacity of ctDNA levels before treatment initiation to predict overall survival (OS) and progression-free survival (PFS) by calculating Harrell’s C-statistic and we compare its predictive value with classical survival surrogates as the pathological response and clinical response assessed according to RECIST criteria v.1.1...Similarly, baseline ctDNA levels predicted OS (c=0.85; 95%CI: 0.72-0.99) better than RECIST criteria (c=0.68; 95%CI: 0.44-0.93). Conclusion Pre-treatment ctDNA levels predicted more accurately long-term survival than radiological assessments in NADIM study and might be useful for the design of new clinical trials.

A positive correlation between the LDH level and cfDNA concentration as well as negative correlation between the LDH level and time to progression was confirmed in a 22-patient cohort. The analysis showed both the potential and limitations of liquid biopsy genetic profiling using HD technology and the Ion Torrent platform.

Implementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.

The assay showed a similar sensitivity to that of the Cobas EGFR Mutation Test v2 at a VAF of 0.5% with 20 ng of input and 100% concordance on clinical samples. The Pan-Cancer Cell-Free Assay can be applied to detect EGFR mutations in advanced lung cancer patients, although follow-up studies will be needed to evaluate the analytical validity for other types of genes and aberrations using clinical samples.

Molecular profiling of liquid biopsies collected upon disease progression using NGS help to identify molecular mechanisms underlying treatment failure.

The cfDNA assay could identify potentially actionable targets for CRC. Identifying how to filter out cancer-like genomic alterations not derived from tumors remains a challenge.

EML4-ALK fusions can be detected at the RNA levels and at the protein levels analyzing EVs derived from H3122 and H2228 cell lines. These results set the stage for the development of EV-based non-invasive ALK testing in NSCLC patients.

Concentrations of baseline ctDNA have been shown to be of prognostic significance.Patients and methods 42 pre-treatment plasma samples from the NADIM clinical trial (NCT03081689), in which resectable stage IIIA NSCLC patients were treated with neoadjuvant chemo-immunotherapy with Nivolumab, were analyzed by NGS, using the Oncomine Pan-Cancer Cell-Free Assay™ (Thermo Fisher Scientific®)...Finally, ctDNA levels at Mutant Allele Frequency (MAF) below 1% at baseline were associated with improved PFS (P=0.025). At 30 months, PFS was 80.30% for these patients compared with 58.33% in patients with ctDNA levels ≥ 1%.Conclusions Molecular profiling of liquid biopsies collected before neoadjuvant chemo-immunotherapy using NGS can identify patients at high risk of progression who might require more aggressive adjuvant treatment in order to achieve a better control of the disease.

The QIAamp Circulating Nucleic Acid Kit was the optimal method for extraction of CSF-ctDNA and the Oncomine cell-free DNA assay is suitable for detection of mutations in CSF-ctDNA. Analysis of CSF-ctDNA is more sensitive than CSF-cytology and has the potential to improve the diagnosis and monitoring of patients with CNS tumors.

"These results demonstrated that combined TNA in the plasma and exoTNA in the pleural fluid can be used to evaluate low-abundant EGFR mutant copies in NSCLC."

This pilot study endorses the use of targeted NGS for non-invasive molecular profiling of breast cancer. Paired sequencing of plasma ctDNA and WBC should be implemented to improve accurate interpretation of liquid biopsy.

Funding: This study was supported by CB16/12/00350 and P118/00226 from ISCIII and Asociación de Mujeres de Apoyo al Cáncer de Mama (AMACMA). AM is recipient of PhD scholarship from Asociación Española Contra el Cáncer (AECC) Valencia Scientific Foundation.