Liquid biopsy in hepatic cancer

808 Background: The impact of BRAF V600E, a known poor prognostic factor in metastatic colorectal cancer (CRC), has not been assessed broadly in non-CRC gastrointestinal (GI) cancers including pancreatic (PC), gastric/gastroesophageal (GC), hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In view of recent tumor-agnostic approval of a BRAF V600E inhibitor, it is important to determine the prevalence and impact of BRAF alterations on non-CRC GI cancer patients (pts). Methods: Genomic results from pts with PC, GC, HCC, or CCA tested with Guardant360 (G360, Guardant Health) as part of clinical care from June 2019 – June 2022 were retrospectively queried. Prevalence of characterized nonsynonymous BRAF genomic alterations (GA) was analyzed; differences between cancer types was assessed using the Chi-squared test. The GuardantINFORM database with aggregated commercial payer health claims and de-identified records from over 243,000 individuals with G360 results was used to derive real-world overall survival (rwOS), reported in months, calculated from diagnosis of metastatic cancer; differences between cancer types were compared using log-rank tests. Results: 23,069 patients were included: PC:49%; GC:24%; CCA:20%; HCC:6%. Pts were more likely to be male (p<0.01). PC/HCC pts were tested more often at new diagnosis (vs at disease progression) than GC/CCA (p=0.02, p<0.01, respectively). BRAF GAs were identified in 2.9%, comparable to tissue-based datasets (MSK-IMPACT: 2.4%); 21% of BRAF GAs were V600E (0.6% prevalence overall). PC/CCA pts were more likely to have BRAF GAs than the overall cohort (p<0.01); they also had more BRAF V600E GAs (p<0.01). rwOS differed by cancer type and BRAF status (Table). Notably, CCA pts with BRAF V600E had superior rwOS compared to CCA pts without BRAF findings (p=0.02). rwOS is not reported for HCC due to small n. Conclusions: Identification of BRAF GAs and V600E in the non-CRC GI population by liquid biopsy is similar to rates observed in tissue-based testing and may be reliably used to assess BRAF status. BRAF GAs and subtypes have mixed prognostic implications on survival for PC/CCA/GC patients that warrants further exploration. [Table: see text]

Introduction The diagnosis of hepatocellular carcinoma (HCC) is made at a relatively advanced stage resulting in poor prognosis. Alpha-fetoprotein and liver ultrasound have limited accuracy as biomarkers in HCC. Liver biopsy provides information on tumor biology; however, it is invasive and holds high threat of tumor seeding. Thus, more accurate and less invasive approaches are needed Areas Covered Highly sensitive liquid biopsy assays have made possible the detection and analysis of cells or organelles such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and tumor-derived exosomes. Here, we focus on CTCs and ctDNA components of liquid biopsy and their clinical application as diagnostic, prognostic, and predictive biomarkers in HCC. Unlike tissue biopsy, liquid biopsy involves attaining a sample at several time frames in an easy and a non-invasive manner. They have been efficacious in detecting and classifying cancer, in predicting treatment response, in monitoring disease relapse and in identifying mechanisms of resistance to targeted therapies. Expert Opinion Although interesting and highly promising, liquid biopsy techniques still have many obstacles to overcome before their wide spread clinical application sees the light. It is expected that these techniques will be incorporated into traditional methodologies for better diagnostic, predictive and prognostic results.

An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumour DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive 'liquid biopsy' for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation marker panel by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumour DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1,098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P < 0.001) and was highly correlated with tumour burden, treatment response, and stage. Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P < 0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.

4110 Background: Need for individualized treatment based on tumor attributes is being reinforced with the rapid development of targeted therapies and immune checkpoint inhibitors for hepatocellular carcinoma (HCC). Lack of mandatory tissue biopsy for the diagnosis of HCC, continues to be a challenge for development of precision medicine for HCC compared to other solid tumors. Liquid biopsy enables comprehensive genomic profiling of circulating tumor DNA (ctDNA) shed from tumors into the circulation. This could compensate for the lack of tissue-based analysis in HCC providing a non-invasive and safer option for genomic profiling. This study aims to address the concordance between commercially available ctDNA and tissue genomic profiling in patients with advanced HCC and to assess the feasibility of liquid biopsy in the treatment of patients with HCC. Methods: Patients subjected to tissue-based next-generation sequencing (NGS) before systemic therapy for advanced HCC at CHA Bundang Medical Center from June 2020 to October 2022 and also had sufficient plasma samples available for ctDNA analysis were included. Oncomine Comprehensive Assay (OCA) ThermoFisher was used for tissue-based NGS and Guardant360 for ctDNA based NGS. For the concordance assessment for the same patient, paired samples of ctDNA and tumor tissue, we investigated the common targeted regions across both the panels. Results: Out of hundred thirty patients included in this study, 98% percent of ctDNA samples for these patients were successfully sequenced; ctDNA were detected in 88% of the samples. For the assessment of concordance between intra-patient ctDNA and tumor tissues, we investigated only clinically important (AMP/ASCO/CAP guidelines tier 1 or 2) variants. When we considered the variants detected in tissues as true positive, the concordance between the mutations detected from ctDNA and tissue samples indicated 68.9% sensitivity and 63.1% positive predictive value (PPV). In ctDNA, targetable alterations were detected in 16.9% of samples, such as ARID1A mutations (7.7%), PTEN mutations (6.9%), PIK3CA hotspot mutations (1.5%), RAS mutations (1.5%), and one RET fusion (0.8%). Regarding the concordance between ctDNA and tumor tissue, the sensitivity for mutation was high for RAS mutation (100.0%), PTEN mutations (100.0%), and ARID1A mutations (75.0%) but low for PIK3CA hotspot mutations (0.0%). One RET fusion was identified only in ctDNA. Conclusions: ctDNA-based genotyping demonstrated clinically acceptable concordance with tissue genomic profiling in patients with advanced HCC. Thus, liquid biopsy using ctDNA help address any challenges associated with the limited use of tissue-based genomic profiling in HCC. Investigation of time between blood and tissue collection and the impact it has on concordance is ongoing.

e16137 Background: Hepatocellular Carcinoma (HCC) is the leading cause of cancer-related mortality in Nigeria. HIV and viral hepatitis B (HBV) and C (HCV) co-infection are common in Nigeria that significantly accelerates liver disease progression including HCC. Aging-related DNA methylation signatures obtained in liquid biopsy, such as circulating cell-free DNA (ccfDNA) extracted from serum/plasma are promising minimally-invasive biomarkers that may inform HIV-associated HCC. We examined the epigenetic age acceleration (EpiAgeAccel) in ccfDNA in HCC patients with HIV. Methods: The study included three groups of participants: a) HIV positive with HCC (n=7); b) HIV positive and cancer-free (n=45); and c) HIV negative with HCC (n=33). Epigenetic age was estimated by Horvath’s calculator using genome-wide ccfDNA methylation data profiled by Illumina EPIC array. EpiAgeAccel was computed as the residuals of a linear model of epigenetic age on chronological age, namely the unexplained portion of epigenetic age by chronological age. We used multiple linear regression to compare EpiAgeAccel between HIV/HCC groups, adjusting for sex, age, education, alcohol intake, and HBV/HCV infection. Results: Among HIV positive participants, there was a higher percentage of men (57% vs. 22%, p&lt;0.001), HBV infection (29% vs. 11%, p=0.004), and HCV infection (57% vs. 2%, p&lt;0.001) in the HCC group compared to the cancer-free group. EpiAgeAccel was 4.8 years higher in HIV positive patients with HCC compared to cancer-free HIV positives (p=0.02). Among HCC patients, EpiAgeAccel was 2.1 years higher in HIV positives compared to HIV negative but not statistically significant. Conclusions: Epigenetic age in ccfDNA is accelerated in HIV-positive HCC patients. EpiAgeAccel measured in ccfDNA may be developed into a surrogate biomarker for minimally invasive HCC detection among HIV-infected patients in low- and middle-income countries.

Purpose: This study aims to develop a liquid biopsy assay to identify HCC and differentially diagnose hepatocellular carcinoma (HCC) from colorectal carcinoma (CRC) liver metastasis. Methods: Thirty-two microRNAs (“HallMark-32” panel) were designed to target the ten cancer hallmarks in HCC. Quantitative PCR and supervised machine learning models were applied to develop an HCC-specific diagnostic model. One hundred thirty-three plasma samples from intermediate-stage HCC patients, colorectal cancer (CRC) patients with liver metastasis, and healthy individuals were examined. Results: Six differentially expressed microRNAs (“Signature-Six” panel) were identified after comparing HCC and healthy individuals. The microRNA miR-221-3p, miR-223-3p, miR-26a-5p, and miR-30c-5p were significantly down-regulated in the plasma of HCC samples, while miR-365a-3p and miR-423-3p were significantly up-regulated. Machine learning models combined with HallMark-32 and Signature-Six panels demonstrated promising performance with an AUC of 0.85-0.96 (p ≤ 0.018) and 0.84-0.93 (p ≤ 0.021), respectively. Further modeling improvement by adjusting sample quality variation in the HallMark-32 panel boosted the accuracy to 95% ± 0.01 and AUC to 0.991 (95% CI 0.96-1, p = 0.001), respectively. Even in alpha fetoprotein (AFP)-negative (< 20ng/mL) HCC samples, HallMark-32 still achieved 100% sensitivity in identifying HCC. The Cancer Genome Atlas (TCGA, n=372) analysis demonstrated a significant association between HallMark-32 and HCC patient survival. Conclusion: To the best of our knowledge, this is the first report to utilize circulating miRNAs and machine learning to differentiate HCC from CRC liver metastasis. In this setting, HallMark-32 and Signature-Six are promising non-invasive tests for HCC differential diagnosis and distinguishing HCC from healthy individuals.

e13034 Background: The Videssa protein-based blood test (Provista Diagnostics) is a combined proteomic biomarker assay that aims to detect established breast cancer – rendering it useful in patients with abnormal or difficult-to-interpret mammograms. Since the incidence of malignancy for BI-RADS 4 biopsies is approximately 20%, a significant percentage of patients undergo needle biopsy unnecessarily. The Videssa assay provides an alternative diagnostic method by way of a non-invasive liquid biopsy. Methods: Our goal was to assess whether this liquid biopsy combined with 3D tomographraphic mammography could accurately predict disease status to reduce the amount of unnecessary tissue biopsies. An IRB-approved, prospective, single-arm study had patients with BI-RADS4 lesions with calcifications requiring tissue biopsy undergo the blood test prior to stereotactic core biopsy. Subsequent results and biopsy pathology were correlated. Results: 46 patients were initially entered with BI-RADS4 calcifications. 9 patients had DCIS (19.6%) and 37 patients had benign calcifications (80.4%). No patient had invasive cancer. Liquid biopsy results were elevated in 2 patients with DCIS (22% sensitivity) and in 5 patients with benign biopsies (10.8%). At interim analysis, the liquid biopsy demonstrated a 28.5% positive predictive value (PPV) and 82% negative predictive value (NPV). Tumor grade did not affect results. We subsequently discontinued using the blood test for BI-RADS4 calcifications and enrolled 13 patients with non-palpable solid lesions to determine the correlation. The liquid biopsy was elevated in 1 of 4 patients with invasive cancer (25% sensitivity) and was not elevated in all 9 patients with benign biopsies (100% NPV). At one-year follow-up, 3 of the 5 patients with an elevated blood assay and negative biopsy have had no incidence of cancer on subsequent imaging. The remaining 2 patients are scheduled for follow-up. Conclusions: In our study, the NPV of the Videssa liquid biopsy is not robust enough to defer tissue biopsy in any patient with indeterminate calcifications on imaging, nor is it specific enough to help predict results in high risk solid lesions. We do not see this liquid biopsy changing our current practice.

This issue of Molecular Oncology focuses on two key topics: blood cancers and liquid biopsy research. We pick the Blood Cancer Awareness month of September to feature the Review Article by Karen Keeshan and colleagues; providing a comprehensive picture of genetic, epigenetic, transcriptional and metabolic profiles of acute myeloid leukemia (AML) in paediatric and adult patients, while also discussing clonality and molecular signatures of stem cells and chemoresistant cells in AML [[1]]. Further Research Articles in this issue investigate specific molecular signatures of AML and other myeloid neoplasms. Indicatively, Andrés-Zayas et al. used a 15-gene panel to identify and characterize myeloid neoplasms with germline predisposition through next-generation sequencing [[2]]. Moreover, Hellesøy et al. observed sex-dependent differences in mutational and transcriptional profiles and ex vivo drug responses of a total of 1755 patients with AML, therefore recommending sex-adjusted clinical management of this heterogeneous disease [[3]]. Liquid biopsy is a valuable tool for diagnosis, patient stratification and treatment follow-up of blood cancers and solid cancers alike, as also reviewed in a recent thematic issue of Molecular Oncology [https://febs.onlinelibrary.wiley.com/toc/18780261/2021/15/6]. Ongoing research aspires to address challenges related to testing sensitivity of liquid biopsy samples. Tyrosine kinase inhibitors (TKIs) are currently being used for the treatment of patients with non-small cell lung cancer (NSCLC) that carry EGFR mutations. However, during the course of treatment, patients often develop resistance to TKI therapies. Thus, mutation profiling of circulating tumour DNA (ctDNA) has been recommended in international guidelines and has entered clinical practice, in order to improve treatment decisions for patients with NSCLC. Unfortunately, ctDNA-based tests show limited sensitivity in detecting EGFR mutations. The research of Luigi Pasini et al. [[4]] proposes the use of RNA contained in blood-derived extracellular vesicles (EVs) for quantitative EGFR profiling in patients with NSCLC subjected to TKI treatment. The study employed longitudinal EGFR mRNA profiling in a representative cohort of patients treated with first- or second-generation TKIs. The authors established a workflow for the ultrasensitive detection of EGFR mutations in circulating EVs and compared the data to previously established ctDNA-based methods. Both methods adequately identified the type of EGFR mutations, where EV-RNA profiling showed superior sensitivity in mutation detection. Additionally, changes in tumour EV burden were observed during disease progression and were associated with clinical features and disease outcomes in patients receiving TKI therapy. The study also suggests that EV-RNA and ctDNA represent independent biological sources in liquid biopsy and that the molecular analysis of both could offer a more comprehensive understanding of tumour dynamics during NSCLC progression. While the role of CTCs in cancer progression, as well as in clinical cancer management, is well established, reaching reproducible and adequate sensitivity of CTC detection and molecular profiling may represent a challenge for cancer diagnostics and treatment selection. Peng-Xiang Wang and colleagues developed a new workflow for CTC enrichment, detection and single-cell analysis [[5]]. The workflow was tested and validated in healthy donors and patients with different types of cancer. In summary, automated CTC enrichment at the ChimeraX-i120 platform was combined with machine learning-based CTC identification and single CTC sequencing. Apart from establishing the novel automated workflow for single CTC enumeration and molecular characterization in different cancer types, the authors employed the workflow in a cohort of patients with hepatocellular carcinoma (HCC) to evaluate the prognostic and clinical feasibility of their method. While the workflow yielded moderate diagnostic potential in distinguishing patients with HCC, it could be efficiently used for an accurate estimation of CTC numbers and as a complementary tool in screening and early diagnosis of patients with different types of cancer. Moreover, single CTC profiling could offer a comprehensive analysis of the tumour clonal composition and tumour heterogeneity before and after treatment. In their article, Bade at al. [[6]] explore a novel strategy aimed at increasing the detection rate of circulating tumour cells (CTCs) in patients with advanced clear cell renal cell carcinoma (ccRCC). Peripheral blood CTCs were isolated and enumerated from a heterogeneous cohort of patients having various disease stages, metastasis sites and types of treatment. Captured CTCs were further characterized for several tumour-specific markers. Analysis of the isolated CTCs demonstrated pronounced heterogeneity of CTC biomarker expression among patients, as well as a significant correlation of CTC numbers with clinical progression. Furthermore, longitudinal CTC characterization showed that CTCs could hold a pharmacodynamic potential for the evaluation of patients during therapy. The novel approach of CTC capture maximized the specificity of CTC identification and advanced the noninvasive evaluation of intra- and intertumoural heterogeneity. The authors propose deployment of these novel assays in prospective clinical trials to explore the clinical potential of their approach in patients with ccRCC. The authors declare no conflict of interest.

Differentiated thyroid cancer (DTC) includes papillary and follicular carcinomas and is the most common type of thyroid cancer. The incidence of this cancer has increased in the last few years, and even if its prognosis is generally good for a subset of patients that does not respond to radioactive iodine (RAI) therapy, the prognosis is much worse: the median overall survival (OS) from discovery of metastasis is 3–5 years and the 10-year survival rate is only 10%. Several mutations, including RAS or RET, as well as BRAF signaling, are associated with thyroid cancer. Liquid biopsy may be useful in selected patient to identify genomic alterations and thus allowing for a precision medicine approach with target therapy. Sorafenib, an oral multi-kinase inhibitor, can be used in the treatment of DTC. Case presentation: A 77 years old. man with diagnosis of metastatic DTC and evidence of presence of mutation of BRAF K601E on liquid biopsy was treated with sorafenib, showing a good response to the treatment and an improvement in the quality of life (QoL). Currently, this patient is still on treatment with sorafenib, gaining control of a multi-metastatic disease, generally characterized by a very poor prognosis. In conclusion, sorafenib has an active role in the treatment of DTC. It also has been considered the standard of care for patients with advanced unresectable hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). In our case we observe the efficacy of using sorafenib in Papillary thyroid carcinoma (PTC) such as confirming both stable disease (SD) in the CT scan as clinical benefit with an increase in QoL. Therefore, use of sorafenib remains an important treatment option, even in case of BRAF mutation, despite a rapidly evolving treatment landscape. It also seems important to perform liquid biopsies, especially in patients in whom it is not possible to repeat a new tissue biopsy. Ongoing clinical trials continue to evaluate sorafenib in different settings, and in combination with other therapies in DTC and HCC.

Hepatocellular carcinoma (HCC), with its high incidence and mortality rate, is one of the most common malignant tumors. Despite recent development of a diagnostic and treatment method, the prognosis of HCC remains poor. Therefore, to provide optimal treatment for each patient with HCC, more precise and effective biomarkers are urgently needed which could facilitate a more detailed individualized decision-making during HCC treatment, including the following; risk assessment, early cancer detection, prediction of treatment or prognostic outcome. In the blood of cancer patients, accumulating evidence about circulating tumor cells and cell-free nucleic acids has suggested their potent clinical utilities as novel biomarker. This concept, so-called “liquid biopsy” is widely known as an alternative approach to cancer tissue biopsy. This method might facilitate a more sensitive diagnosis and better decision-making by obtaining genetic and epigenetic aberrations that are closely associated with cancer initiation and progression. In this article, we review recent developments based on the available literature on both circulating tumor cells and cell-free nucleic acids in cancer patients, especially focusing on Hepatocellular carcinoma.

Cancer ranks among the most lethal diseases worldwide. Tissue biopsy is currently the primary method for the diagnosis and biological analysis of various solid tumors. However, this method has some disadvantages related to insufficient tissue specimen collection and intratumoral heterogeneity. Liquid biopsy is a noninvasive approach for identifying cancer-related biomarkers in peripheral blood, which allows for repetitive sampling across multiple time points. In the field of liquid biopsy, representative biomarkers include circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes. Many studies have evaluated the prognostic and predictive roles of CTCs and ctDNA in various solid tumors. Although these studies have limitations, the results of most studies appear to consistently demonstrate the correlations of high CTC counts and ctDNA mutations with lower survival rates in cancer patients. Similarly, a reduction in CTC counts throughout therapy may be a potential prognostic indicator related to treatment response in advanced cancer patients. Moreover, the biochemical characteristics of CTCs and ctDNA can provide information about tumor biology as well as resistance mechanisms against targeted therapy. This review discusses the current clinical applications of liquid biopsy in cancer patients, emphasizing its possible utility in outcome prediction and treatment decision-making.

Retraction The abstract by Raimondi et al entitled, “Resistance to CDK4/6 inhibitors (CDK4/6i): The clinical usefulness of liquid biopsy in metastatic breast cancer (mBC),” published in Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021) 1053-1053, was retracted by the Journal of Clinical Oncology. ( JCO). An Expression of Concern was previously issued by JCO on June 16, 2021. Subsequent to publication of the abstract, questions about the data were brought to JCO’s attention. The data were provided by author Giuseppe Naso, who has since died. As the source of the data and methods of data collection cannot be verified, the abstract is being retracted. A copy of this Retraction Notice was sent to the last known email addresses for the authors. Authors Lucrezia Raimondi, Laura Giaconi, and Gian Paolo Spinelli agreed to the retraction. Authors Rachele Lazzeroni, Laura Di Benedetto, Filippo Maria Raimondi, and Arianna Di Rocco did not respond to our queries. Author Giuseppe Naso is deceased. This abstract was retracted on November 9, 2021. 1053 Expression of Concern The abstract by Raimondi et al entitled, “Resistance to CDK4/6 inhibitors (CDK4/6i): The clinical usefulness of liquid biopsy in metastatic breast cancer (mBC),” published in Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021) 1053-1053, is under further review. Questions have been raised regarding the integrity of the methods, results, and analysis of the reported abstract. Until the authors and their institutions can fully provide additional information, readers should interpret the findings presented with caution. An update will be provided when our investigation is complete. Background: Palbociclib (P) in combination with fulvestrant (F) or letrozole (L), is used globally to treat metastatic breast cancer but despite therapeutic improvements most patients acquire resistance to CDK4/6i. KRAS tumor mutations (mutKRAS) have been associated with worse PFS in several tumor types but have not been analysed extensively in breast cancer. To understand the molecular mechanisms of resistance to CDK4/6i and their clinical behavior, using liquid biopsy, we evaluated the opportunity to reveal the onset of resistance to CDK4/6i detecting mutKRAS ctDNA. Methods: We studied the KRAS mutation status of 211 patients with mBC treated with CDK4/6i plus L or F as first-line metastatic therapy. Using Bio-Rad QX200 droplet digital polymerase chain reaction (ddPCR) system we determined KRAS ctDNA levels in plasma. Using logistic and Cox regression, a predictive model for objective response (OR), progression-free survival (PFS) and overall survival (OS) was constructed. The PFS and the OS were estimated by the Kaplan–Meier method and compared with use of the log-rank test. Results: In 38% (81 patients, 24 in treatment with L and 57 in treatment with F) we observed mutKRAS ctDNA before starting CDK4/6i: the detection of mutKRAS significantly correlated with the onset of resistance to CDK4/6i within 6months from the evidence of KRAS mutation and worse PFS ( p&lt;0.001). OR was seen in 84 of 130 KRAS wild-type (WT) patients versus 0 of 81 in KRAS mutants. At 24-month follow up, median PFS was significantly better in KRAS WT versus mutants (3.1 [range: 1-6months,95%CI 0.9-3.6] versus NA months; p&lt;0.001). Correlating the results of liquid biopsy both to tumoral burden and patients clinical features, we observed a higher mutKRAS circulating copies-number in those patients with two or more metastatic sites( p&lt;0.001). Conclusions: Despite the study’s limitations, our data suggest mutKRAS ctDNA status leads to CDK4/6i resistance acquisition within 6 months from the detection and provide critical information for the prediction of therapeutic responses in mBC. Monitoring KRAS status with liquid biopsy, we could predict who will take advantage from CDK4/6i, decreasing wastes of resources ensuring the best patients’ quality of life.

Personalized therapy for hepatocellular carcinoma: Where are we now?

Background In the era of personalized therapy liquid biopsy is considered an important diagnostic tool in the clinical management of cancer patients. Tissue specimen represents “gold standard” for molecular evaluation of specific gene targets alterations that lead cancer patients to benefit of a “tailed therapy” based on molecular features of the tumor. This innovative source of nucleic acids was introduced in clinical setting only for NSCLC patients to test Epidermal Grow Factor Receptor (EGFR) mutations when tissue is not available or to monitor acquired resistance mutation after a first line of treatment. The study aimed at assessing the diagnostic potential of liquid biopsy in balanced tertiary screening modeling. Methods The cases relating to 5 years of activity regarding to molecular diagnostics performed on liquid biopsy specimens in the Predictive Diagnostic laboratory of AOU Federico II were reviewed. Laboratory data were collected in SPSS. Non parametric analysis were performed in order to test the differences between patients WILD TYPE or not. A multivariate logistic model was performed in order to assess the effect of mutation, age and sex, on the tumor progression. The results of the revision concern 515 total cases (almost of all plasma or peripheral blood) allowed to evaluate the liquid biopsies for women and men. The average age of the Patients is 66.3 years, and the 25 percentile is 59 years. Results The cases are 221 basal and 294 by progression. The cases with mutation, as expected, have an OR 4,15 compared to the basal to have a tumor progression (95% IC: 2,7 - 6,3) regardless of sex and age. The mutations detected were 131 from different types of pulmonary carcinomas. Conclusions Working on case data, specifying the characteristics of the Patients with mutations will drive a further estimate in tertiary prevention screening designs. Key messages In the last five years, the liquid biopsy has been used for different purposes in oncology. To specify the characteristics of the Patients with mutations will drive a further estimate in tertiary prevention screening designs.

Liquid biopsy in lung cancer: present and futureIn lung cancer, as well as in other malignancies, the so-called "liquid biopsy" is quickly moving from research into clinical practice.Although it has not yet reached its full potential, the "liquid biopsy" is no longer a promise but a reality that is allowing a better monitoring and treatment selection of cancer patients in many hospitals and oncology departments.We can already envision a day when "liquid biopsy" tests will be as common and useful as imaging techniques and "standard" biopsies, offering invaluable prognostic and predictive information.The objective of this special issue of Translational Lung Cancer Research (TLCR) is to offer an overview of this exciting area, with a particular emphasis on the clinical utility of the so-called "liquid biopsy" and the diversity of applications, methodologies and information that can be derived from it."Liquid biopsy" refers to non-invasive tests done in blood samples (or other body fluids) from cancer patients in order to detect materials originated in the tumor.Although the term is almost universally used, many pathologists argue that is not correct.In their opinion, the so-called "liquid biopsies" are not true biopsies.These pathologists are probably right.A true biopsy is usually performed by a surgeon or a pneumologist and involves extraction of tissues or sample cells for examination by a pathologist under a microscope, commonly after some kind of fixation and staining."Liquid biopsies" are not obtained by surgeons, involve extraction of blood and not of solid tissues, pathologists only occasionally intervene and fixation or staining are equally rare.To make things worse, the "liquid" part in the term "liquid biopsy" can also be misleading.The materials originated in the tumor that are to be detected in such a "biopsy" are never liquids.Sometimes they are cells or fragments of cells, such as circulating-tumor cells (CTCs), exosomes or tumor-educated platelets; sometimes they are nucleic acids dissolved in the blood, such as circulating tumor DNA or RNA (ctDNA, ctRNA).The differences between a "real" and a "liquid" biopsy or "liquid sample", as the pathologists would probably prefer to call them, explain the advantages of the latter."Liquid" biopsies will never replace real biopsies, which are irreplaceable sources of information that cannot be obtained by any other means.However, they are already offering all sorts of additional data that could not be obtained in any other way.In patients who cannot be biopsied, or where biopsies do not have enough tissue, "liquid biopsy" offers a mean to perform genetic testing for targeted therapy.Also, in patients with advanced disease, it is not feasible to obtain biopsies of every metastatic site.But blood reaches both the primary tumor and the metastases, and materials coming from all can be found in a "liquid biopsy".Finally, unlike "real" biopsies, blood can be repeatedly obtained and used to monitor the course of the disease, including early detection of response and relapse or emergence of resistance to a particular therapy.In the first article of this special issue, Liquid biopsy based biomarkers in non-small cell lung cancer for diagnosis and treatment monitoring, Pérez-Callejo et al. review the methodologies available for the isolation and analysis of ctDNA, exosomes, tumoreducated platelets and CTCs, describe the unique information that can be derived from each of these materials and discuss their clinical applications in lung cancer.Their conclusion is clear; liquid biopsies, particularly CTCs and cfDNA, will guide treatment decision, improve the outcome for lung cancer patients and allow early diagnosis of tumors that are not yet visible on imaging.CTCs and ctDNA are the two materials most commonly analyzed in lung cancer "liquid biopsies".Which one should be preferred is a burning issue that is discussed by Calabuig-Fariñas et al. in their article Circulating tumor cells versus circulating tumor DNA in lung cancer.Which one will win?Their answer is that this will be a war with two victors, since CTCs and ctDNA will play complementary roles based on their relative strengths and limitations.They consider that ctDNA will be the material chosen for the analysis of mutations, copy number aberrations and DNA methylation changes, while CTCs, which provide the opportunity to study whole cells, will allow DNA, RNA, and protein-based molecular profiling, in addition to in vivo studies.If CTCs and ctDNA are already a reality in the clinical practice, exosomes constitute one of the most promising areas of research.In this special issue, they are reviewed in the article Exosomes as a source of genetic material in non-small cell lung cancer: a truly Pandora's box.There, Reclusa et al. prove the accuracy of this definition.The stability of the exosomes in the blood and their similarity to the cells of origin, make it possible to detect in them all types of biomarkers as well as the "instructions" for migration and aggressiveness of the tumor and information on druggable targets.In view of this, once methodologies are properly standardized, the authors conclude that exosome analysis will be quickly incorporated in the clinical practice.