Molecular Diagnosis Of Cancer Research Articles

Basic knowledge for counseling patients undergoing risk-reducing salpingo-oophorectomy.

Significant progress has been made in the molecular diagnosis of cancer. It provides personalized medicine, including cancer diagnosis, prognosis, targeted therapy, and risk detection. These advances allow physicians to identify patients at risk for cancer before it develops and offer them an opportunity to prevent its development. Mutations in breast cancer susceptibility genes 1 and 2 (BRCA1 and 2) are one of the most well-known cancer-related gene mutations since actor Angelina Jolie shared her experience with genetic mutations and risk-reducing surgery in the media. In Korea, tests for germline BRCA1/2 mutations have been covered by insurance since May 2012 and the number of women of BRCA1/2 mutations has continued to increase over the past decade. Most carriers of BRCA1/2 mutations consider risk-reducing salpingo-oophorectomy (RRSO) resulting in early menopause and want to know the lifetime risks and benefits of RRSO. However, despite the increasing number of carriers of BRCA1/2 mutations, the counseling and management of patients requiring RRSO varies among physicians. This article provides basic knowledge on RRSO to help physicians comprehensively assess its risks and benefits and manage at-risk women.

Comparison between next-generation sequencing and multiplex polymerase chain reaction assays for nonsmall-cell lung cancer molecular diagnosis.

Genetic mutation detection has become an important step in nonsmall-cell lung cancer (NSCLC) treatment because of the increasing number of drugs that target genomic rearrangements. A multiplex test that can detect multiple gene mutations prior to treatment is thus necessary. Currently, either next-generation sequencing (NGS)-based or polymerase chain reaction (PCR)-based tests are used. We evaluated the performance of the Oncomine Dx Target Test (ODxTT), an NGS-based multiplex biomarker panel test, and the AmoyDx Pan Lung Cancer PCR Panel (AmoyDx PLC panel), a real-time PCR-based multiplex biomarker panel test. Patients with histologically diagnosed NSCLC and a sufficient sample volume to simultaneously perform the AmoyDx PLC panel and ODxTT-M were included in the study. The success and detection rates of both tests were evaluated. Biopsies revealed 116 cases of malignancies, 100 of which were NSCLC. Of these, 59 met the inclusion criteria and were eligible for analysis. The success rates were 100% and 98% for AmoyDx PLC panel and ODxTT-M, respectively. Nine driver mutations were detected in 35.9% and 37.3% of AmoyDx PLC and ODxTT-M panels, respectively. EGFR mutations were detected in 14% and 12% of samples using the AmoyDx PLC panel and ODxTT-M, respectively. Of the 58 cases in which both NGS and AmoyDx PLC panels were successful, discordant results were observed in seven cases. These differences were mainly due to different sensitivities of the detection methods used and the gene variants targeted in each test. The AmoyDx PLC panel, a PCR-based multiplex diagnostic test, exhibits a high success rate. The frequency of the nine genes targeted for treatment detected by the AmoyDx PLC panel was comparable to the frequency of mutations detected by ODxTT-M. Clinicians should understand and use the AmoyDx PLC panel and ODxTT-M with respect to their respective performances and limitations.

A spatially-controlled DNA triangular prism nanomachine for AND-gated intracellular imaging of ATP in acidic microenvironment.

ADNA triangular prism nanomachine (TPN)-based logic device for intracellular AND-gated imaging of adenosine triphosphate (ATP)has been constructed. By using i-motif sequences and ATP-binding aptamers as logic control units, the TPN logic device is qualified to respond to the acidic environment and ATP in cancer cell lysosomes. Once internalized into the lysosome, the specific acidic microenvironment in lysosome causes the i-motif sequence to fold into a tetramer, resulting in compression of DNA tri-prism. Subsequently, the split ATP aptamer located at the tip of the collapsed triangular prism binds stably to ATP, which results in the fluorescent dyes (Cy3 and Cy5) modified at the ends of the split aptamer being in close proximity to each other, allowing Förster Resonance Energy Transfer (FRET) to occur. The FRET signals are excited at a wavelength of 543 nm and can be collected within the emission range of 646-730 nm. This enables the precise imaging of ATP within a cell. We also dynamically operate AND logic gates in living cells by modulating intracellular pH and ATP levels with the help of external drugs. Owing to the AND logic unit on TPN itcan simultaneously recognize two targets and give corresponding intelligent logic judgment via imaging signal output. Theaccuracy of molecular diagnosis of cancer can be improved thuseliminating the false positive signal of single target-based detection. Hence, this space-controlled TPN-based logical sensing platform greatly avoids sensitivity to extracellular targets during the cell entry process, providing a useful tool for high-precision imaging of the cancer cell's endogenous target ATP.

Advanced technologies for molecular diagnosis of cancer: State of pre-clinical tumor-derived exosome liquid biopsies.

Exosomes are membrane-defined extracellular vesicles (EVs) approximately 40-160​nm in diameter that are found in all body fluids including blood, urine, and saliva. They act as important vehicles for intercellular communication between both local and distant cells and can serve as circulating biomarkers for disease diagnosis and prognosis. Exosomes play a key role in tumor metastasis, are abundant in biofluids, and stabilize biomarkers they carry, and thus can improve cancer detection, treatment monitoring, and cancer staging/prognosis. Despite their clinical potential, lack of sensitive/specific biomarkers and sensitive isolation/enrichment and analytical technologies has posed a barrier to clinical translation of exosomes. This review presents a critical overview of technologies now being used to detect tumor-derived exosome (TDE) biomarkers in clinical specimens that have potential for clinical translation.

Brazilian Expert Consensus for NTRK Gene Fusion Testing in Solid Tumors.

Oncogenic neurotrophic tropomyosin receptor kinase gene fusions occur in less than 1% of common cancers. These mutations have emerged as new biomarkers in cancer genomic profiling with the approval of selective drugs against tropomyosin receptor kinase fusion proteins. Nevertheless, the optimal pathways and diagnostic platforms for this biomarker's screening and genomic profiling have not been defined and remain a subject of debate. A panel of national experts in molecular cancer diagnosis and treatment was convened by videoconference and suggested topics to be addressed in the literature review. The authors proposed a testing algorithm for oncogenic neurotrophic tropomyosin receptor kinase gene fusion screening and diagnosis for the Brazilian health system. This review aims to discuss the latest literature evidence and international consensus on neurotrophic tropomyosin receptor kinase gene fusion diagnosis to devise clinical guidelines for testing this biomarker. We propose an algorithm in which testing for this biomarker should be requested to diagnose advanced metastatic tumors without known driver mutations. In this strategy, Immunohistochemistry should be used as a screening test followed by confirmatory next-generation sequencing in immunohistochemistry-positive cases.

Innovative Diagnostic Peptide-Based Technologies for Cancer Diagnosis: Focus on EGFR-Targeting Peptides.

Active targeting using biological ligands has emerged as a novel strategy for the targeted delivery of diagnostic agents to tumor cells. Conjugating functional targeting moieties with diagnostic probes can increase their accumulation in tumor cells and tissues, enhancing signal detection and, thus, the sensitivity of diagnosis. Due to their small size, ease of chemical synthesis and site-specific modification, high tissue penetration, low immunogenicity, rapid blood clearance, low cost, and biosafety, peptides offer several advantages over antibodies and proteins in diagnostic applications. Epidermal growth factor receptor (EGFR) is one of the most promising cancer biomarkers for actively targeting diagnostic and therapeutic agents to tumor cells due to its active involvement and overexpression in various cancers. Several peptides for EGFR-targeting have been identified in the last decades, which have been obtained by multiple means including derivation from natural proteins, phage display screening, positional scanning synthetic combinatorial library, and in silico screening. Many studies have used these peptides as a targeting moiety for diagnosing different cancers in vitro, in vivo, and in clinical trials. This review summarizes the progress of EGFR-targeting peptide-based assays in the molecular diagnosis of cancer.

Detection of BRCA1/2 pathogenic variants in patients with breast and/or ovarian cancer and their families. Analysis of 3,458 cases from Lower Silesia (Poland) according to the diagnostic algorithm of the National Cancer Control Programme.

Breast and ovarian cancers are among the most common malignancies in the female population, with approximately 5–10% of cases being hereditary. BRCA1 and BRCA2 with other homologous recombination genes are the most tested genes in hereditary breast and ovarian cancer (HBOC) patients. As next-generation sequencing (NGS) has become a standard and popular technique, such as for HBOC, it has greatly simplified and accelerated molecular diagnosis of cancer. The study group included 3,458 HBOC patients or their relatives from Lower Silesia (Poland) (a voivodeship located in south-west Poland inhabited by 2.9 million people). All patients were tested according to the recommendations from the National Cancer Control Programme of the Ministry of Health for the years 2018–21. We tested 3,400 patients for recurrent pathogenic variants for the Polish population: five BRCA1 founder variants (c.5266dup, c.181T>G, c.4035del, c.3700_3704del, and c.68_69del), two PALB2 variants (c.509_510del, c.172_175del) and three CHEK2 variants [c.1100del, c.444+1G>A, g.27417113-27422508del (del5395)]. Next 260 patients from the study group were chosen for the BRCA1/2 NGS panel, and additionally selected marker pathogenic variants were tested using Sanger sequencing and MLPA methods in 45 and 13 individuals, respectively. The analysis of BRCA1/2 in the 3,458 patients with HBOC or their relatives revealed 144 carriers of 37 different pathogenic variants (22 in BRCA1 and 15 in BRCA2). Among all detected variants, 71.53% constituted founder pathogenic BRCA1 variants. Our study has revealed that for the Lower Silesian population, the first-line BRCA1/2 molecular test may be limited to only three variants in BRCA1—c.5266dup, c.181T>G, and c.4035del—but the aim should be to provide a full screening test of HBOC critical genes. The key and still growing role of molecular diagnostics of neoplasms, which includes HBOC, is undeniable. Therefore, it is necessary to provide complete and optimal therapeutic and prophylactic algorithms in line with current medical knowledge.

Tumor-Derived Exosomal RNA From Fine-Needle Aspiration Supernatant as a Novel Liquid Biopsy for Molecular Diagnosis of Cancer.

Background: We hypothesized that the fine needle aspiration (FNA) supernatant from tumor might contain tumor-derived exosomes. The objective of this pilot study was to test if tumor-derived exosomal RNA could be found in FNA supernatants for molecular diagnosis of cancer. Methods: 10 FNA samples from pancreatic tumor were included. After the routine recuperation of cellular material by centrifugation, the cell-free Cytolyt liquid was collected instead of being discarded. 10 ml Cytolyt was used to isolate the exosomes. Transmission electronic microscopy (TEM) was used to examine the presence of exosomes. The exosomal marker CD63 was analyzed by flow cytometry. The exosomal RNA was extracted. RT-qPCR was performed to detect the GAPDH and the tumor marker of glypican 1 gene expression. Results: TEM confirmed the presence of exosomes from FNA supernatants. Flow cytometry showed a strong positive expression of exosome marker CD63. The concentration of exosomal RNA ranged from 18.81 to 354.75 ng/μl with an average of 81.76 ng/μl. The average exosomal RNA quantity was 1390.01 ng (range from 319.77 to 6030.75 ng) with an average 260/280 ratio of 2.12. GAPDH was detectable in all samples. Exosomal glypican 1 was detected in all samples of pancreatic ductal adenorcarcinomas (3/3) and absent from benign cystic samples (3/3). Furthermore, exosomal glypican 1 was positive in one sample with a non-contributive cytology and in one sample in which no malignant cell was found. Conclusion: This is the first report that the supernatants from FNA biopsy may contain tumor-derived exosomal RNA. These tumor-derived exosomes from FNA may provide a new liquid biopsy for the molecular diagnosis of cancer.

Hydrogel Stamping for Rapid, Multiplexed, Point-of-Care Immunostaining of Cells and Tissues.

In the era of precision oncology, multicolor fluorescence imaging has become a core technology for multiplexed molecular analysis of cellular and tissue specimens. However, conventional solution-based staining is labor-intensive and time-consuming and requires considerable expertise to yield optimal results, which creates difficulties for employing this technology in resource-limited settings. Here, we report a new immunostaining method based on hydrogel stamping, which is simple, fast, easy to use, and reproducible. We showed that a hydrophilic hydrogel stamp could effectively transfer fluorescent antibodies to targets and withdraw an excess solution when the reaction is completed, obviating the need for extra washing. This unique property allows for quality immunostaining in 5 min for cells using one-eighth of antibody consumption compared to the conventional solution-based method. Furthermore, we implemented fluorescence quenching and immunocycling with hydrogel staining for multiplexed analysis of 9 protein markers at a single cell level. Finally, we applied the immunocycling method to human breast cancer tissue samples and showed quality immunostaining over a large area (∼2 cm2) in 30 min for molecular subtyping of breast cancer. The hydrogel immunostaining could open new opportunities for rapid, automated, and multiplexed profiling in compact point-of-care systems for molecular cancer diagnosis.

When blockchain meets artificial intelligence: An application to cancer histopathology

A recent study by Saldanha et al. demonstrates that blockchain-based models outcompeted local models and performed similarly with merged models to predict molecular features from cancer histopathology images. The results reveal the capability of decentralized models in molecular diagnosis of cancer.

Nanotechnology in Tumor Biomarker Detection: The Potential of Liganded Nanoclusters as Nonlinear Optical Contrast Agents for Molecular Diagnostics of Cancer.

Simple SummaryThe key factor in preventing premature death from cancer is an early and accurate diagnosis. While common diagnostic procedures are successful in the detection and rough description of a tumor mass, a deeper insight into cancer’s molecular features is needed to optimize the treatment and increase the chances of survival. Nanotechnology can aid the molecular diagnostics of cancers through a design of nanomaterials that can simultaneously recognize specific cancer-associated molecules, so-called tumor biomarkers, and allow for their visualization by different imaging techniques. With a recent explosion in the production of various nanomaterials, the selection of the most suitable nanomaterial for tumor biomarker detection becomes a challenge. In this article, we review recent advances in the molecular diagnostics of cancer using nanotechnology and focus on liganded noble metal quantum nanoclusters, a class of ultrasmall nanomaterials with distinctive structural and optical properties, as tools in tumor biomarker detection.Cancer is one of the leading causes of premature death, and, as such, it can be prevented by developing strategies for early and accurate diagnosis. Cancer diagnostics has evolved from the macroscopic detection of malignant tissues to the fine analysis of tumor biomarkers using personalized medicine approaches. Recently, various nanomaterials have been introduced into the molecular diagnostics of cancer. This has resulted in a number of tumor biomarkers that have been detected in vitro and in vivo using nanodevices and corresponding imaging techniques. Atomically precise ligand-protected noble metal quantum nanoclusters represent an interesting class of nanomaterials with a great potential for the detection of tumor biomarkers. They are characterized by high biocompatibility, low toxicity, and suitability for controlled functionalization with moieties specifically recognizing tumor biomarkers. Their non-linear optical properties are of particular importance as they enable the visualization of nanocluster-labeled tumor biomarkers using non-linear optical techniques such as two-photon-excited fluorescence and second harmonic generation. This article reviews liganded nanoclusters among the different nanomaterials used for molecular cancer diagnosis and the relevance of this new class of nanomaterials as non-linear optical probe and contrast agents.

RHAMM-Target Peptides as Molecular Imaging Probes for the Imaging of RHAMM-Expressing Cancer Cells

The incidence of cancer in the world is growing steadily. Therefore, it is necessary to develop new approaches for the early diagnosis of cancer. This work is devoted to the study of the potential of RHAMM-target peptides for molecular diagnosis of cancer. The key amino acids of the RHAMM target peptides were identified by the alanine scan method. The specificity of binding of peptides to RHAMM-CT was assessed using competitive HA substitution by the ELISA method. RHAMM-CT was obtained by genetic engineering and isolated by affinity chromatography. The interaction of RHAMM target peptides with the surface receptor of tumor cells was evaluated by confocal microscopy. It has been shown that fragment EEGEEZ in the peptides' composition is necessary for binding to the RHAMM-CT. The results showed that the RHAMM-target peptides bind specifically to the RHAMM-CT and competitively substituted HA at the RHAMM. It has been found that aggrecan is unable to displace peptides from the HA binding site of RHAMM-CT. The results showed that the FITC peptide binds specifically to RHAMM on the surface of prostate cancer cells. Therefore, RHAMM-target peptides have the potential for early molecular diagnosis of cancer.

Comprehensive and Computable Molecular Diagnostic Panel (C2Dx) From Small Volume Specimens for Precision Oncology: Molecular Subtyping of Non-Small Cell Lung Cancer From Fine Needle Aspirates.

The Comprehensive, Computable NanoString Diagnostic gene panel (C2Dx) is a promising solution to address the need for a molecular pathological research and diagnostic tool for precision oncology utilizing small volume tumor specimens. We translate subtyping-related gene expression patterns of Non-Small Cell Lung Cancer (NSCLC) derived from public transcriptomic data which establish a highly robust and accurate subtyping system. The C2Dx demonstrates supreme performance on the NanoString platform using microgram-level FNA samples and has excellent portability to frozen tissues and RNA-Seq transcriptomic data. This workflow shows great potential for research and the clinical practice of cancer molecular diagnosis.

P37.25 Clinical Validation of Low-costs Next Generation Sequencing Panels in Solid Tumors

The implementation of personalized medicine has revolutionized the current strategies in oncology. Numerous comparisons of technology platforms for mutation testing conclude that the methods should offer high sensitivity, tissue economy and short turnaround time for physicians to guide treatment decisions. In the current study, we were trying to validate the low cost NGS panels in different solid tumors. In addition, validation sample tumors include lung cancers, colorectal cancers and thyroid cancers from both Taiwan and Vietnam. A total of 180 tumor samples were retrospectively collected from Cardinal Tien Hospital (Taiwan) and Bach Mai Hospital (Vietnam) between 2015∼2019. Thirty EGFR mutation positive non-small cell lung cancer (NSCLC) samples, thirty KRAS/NRAS mutation positive colorectal cancer (CRC) samples, and thirty BRAF mutation positive thyroid cancer (TC) samples were chosen from each hospital. All the mutations were tested by Sanger sequencing and NGS. There were 38 EGFR L858R mutation cases, 22 EGFR 19 deletion mutation cases, 2 EGFR T790M mutation cases, 29 KRAS G12D mutation cases, 24 KRAS G12V mutation cases, 5 KRAS G13C mutation cases, 2 NRAS G12S mutation cases, and 60 BRAF V600E mutation cases. The 2 EGFR T790M cases were also positive for the EGFR L858R mutation. The results were the same with Sanger sequencing which give the sensitivity, specificity, positive predict value and negative predict value to be 100%. The low cost RaSeqTM molecular testing system is an accessible, rapid, and reliable testing option for both routine and challenging FFPE specimens. It can serve well as a routine molecular diagnostic tool in most pathology laboratories. The high sensitivity and high accuracy with different gene tests simultaneously are the most advantage in the clinical cancer molecular diagnosis.

Abstract IA08: Incorporating proteomics in bladder cancer molecular diagnosis

Abstract Proteins are directly linked to cell phenotype, hence accrue interest as biomarkers and therapeutic targets. Investigating the urine proteome as a source of biomarkers entails multiple technical challenges, with the main being the protein concentration vast dynamic range and presence of proteins in multiple modified forms. As a result, protein enrichment strategies in combination to different mass spectrometry platforms are regularly employed as main discovery tools, providing complementary experimental outputs. Verification and further biomarker validation regularly involve application of ELISA and, more recently, multiplex mass spectrometry (MS)-based assays (Multiple Reaction Monitoring–MS). In the course of this presentation, an overview of urine proteomics, as applied in bladder cancer research, will be provided. In brief, using the armamentarium of existing methodologic approaches, multiple urinary proteins have been associated with bladder cancer phenotypes and/or attributed prognostic or predictive value, including the Nuclear Matrix Protein 22 (NMP22) and Bladder Tumor Antigen (BTA; corresponding to complement factor H-related protein), approved by the FDA for use in disease diagnosis and/or surveillance. Additional proteins frequently reported in the literature as associated with bladder cancer detection and/or staging include various forms of apolipoproteins, proteins of the extracellular matrix (such as collagen and fibrinogen fragments), matrix metalloproteinases, cytokeratin fragments as well as proteins involved in apoptosis (such as survivin) or inflammation (including multiple cytokines). Reports also support potential of some of these proteins when evaluated in the form of biomarker panels, as predictors of BCG response in high-risk non-muscle invasive bladder cancer. Along these lines, simultaneous analysis of multiple markers through ELISA or mass spectrometry-based platforms has provided increased accuracies in comparison to single biomarkers. Hematuria is a frequent confounding variable in urinary protein measurements, rendering establishment of signal specificity via optimized ELISA assays or mass spectrometry platforms a current technological target. Direct links of urinary protein changes to bladder tissue events have been frequently reported (such as the cases of profilin 1 or activated leukocyte cell adhesion molecule). Even though disproportionate to the numerous reported findings (frequently compromised by suboptimal experimental design), collectively, promising urinary protein findings in association to bladder cancer phenotypes and contexts of use are existent, awaiting proper validation in large-scale prospective trials. Main research paths that now open up include: refinement of existing bladder cancer molecular subtypes, with the addition of information at the protein level, and placing urinary markers in this context; and, in parallel, design and running of proper validation studies as well as clinical sample sharing, to define optimal marker combinations per clinically relevant bladder cancer context of use. In the latter, special emphasis should be placed on prediction of treatment response, in order to maximize benefit from available treatment modalities. Citation Format: Antonia Vlahou. Incorporating proteomics in bladder cancer molecular diagnosis [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr IA08.

Stimulus-responsive surface-enhanced Raman scattering: a "Trojan horse" strategy for precision molecular diagnosis of cancer.

Molecular diagnosis has played an increasingly important role in cancer detection. However, it remains challenging to develop an in situ analytical method capable of profiling the molecular phenotype of tumors for precision cancer diagnosis. A “Trojan horse” strategy based on stimulus-responsive surface-enhanced Raman scattering (SR-SERS) is reported here for selectively recording the comprehensive molecular information of tumors in situ, without resorting to destructive sample preparation and complex data analysis. This technique is employed to delineate the margin between tumors and normal tissues with high accuracy, and to further discriminate the molecular fingerprints of tumors in the early and late stages. Based on molecular profiling, we discovered that the signal ratios of fatty acid-to-phenylalanine could serve as promising indicators for identifying the primary tumors in different stages. This simple SR-SERS technique also provides a potential useful means for identifying tumor classifications or distinguishing primary and metastatic tumors.

Fast Cancer Molecular Diagnosis from FFPE Tissues Based on Metabolic Profiles Using SpiderMass Technology

Formalin-fixation and paraffin-embedding (FFPE) is the worldwide gold standard for tissue preservation and routine material for general diagnostics and cancer in particular. Despite the fact that histology is complemented by other modalities such as in situ hybridization (ISH) or immunohistochemistry (IHC), the pathology can fail to provide the right diagnosis in a certain number of cases. Overall, the standard process is time-consuming, labor intensive and doesn’t reach the right sensitivity and specificity. Lipids are central players in cancer. Unfortunately, to this date lipids aren’t used for diagnostics and lipid analysis from FFPE has never been reported due to tissue processing which limits their detection. Here, we report a new method for cancer diagnostics based on their unique tissue-lipidomic signatures through rapid molecular screening of FFPE sections without dewaxing using Water-Assisted Laser Desorption Ionization Mass Spectrometry (WALDI-MS) - SpiderMass. Advantageously, the method does not require human intervention. The annotated tissues are first analyzed to train the system and then blind samples can be diagnosed in less than a few minutes. The platform was demonstrated for the diagnostics of canine and human sarcoma, including cases which are known to be problematic in the gold standard workflow. Interrogation of blind samples against the built models revealed over 95% probability values to discriminate specific subtypes

ES18.02 When to Switch to a New TKI : Imaging Based Crtieria or Positive Liquid Biopsy?

The discovery of specific mutations and associated addicted pathways in lung adenocarcinoma cells has led to the development of many targeted therapies useful for corresponding activating mutation. In addition, conventional chemotherapy and novel immunotherapy such as PD1/PDL1 antibodies are also very effective for lung cancer patients with or without specific driver mutation. Patients with metastatic lung cancer nowadays can be treated with multiple lines of effective treatment. Patients who receive more lines of therapy tend to live longer than those who had only receive limited number of treatment. In order to maximize the overall survival outcome of metastatic lung cancer patient who receives systemic treatment, it is important to find out the best first line choices and the best timing to change the treatment regimen to second line and so forth. Tumor reduction or stabilization by imaging criteria has been used widely for a long time in chemotherapy and targeted therapy era. RECIST (Response evaluation criteria in solid tumors) has been adopted as a uniform criteria for cancer clinical trial to classify the tumor response to certain treatment. However, in order to accommodate different cancer types and sites of metastasis, several variants of RECIST emerged to better evaluate the treatment response and necessity of maintain the present treatment. In daily practice, changes of tumor size in the image provide the best guidance for clinicians to continue or change regimen for the patients. In patients who has no reliable evaluable radiological image for follow up, such as patients who presented with effusion, bone metastasis, leptomeningeal metastasis or tumor with poor margin, patients’ clinical performance and alterations in tumor markers occasionally can provide clinicians information for judgement. The introduction of cell free plasma tumor DNA for the molecular diagnosis of cancer gave us a new change of how to manage the patients properly when changing regimen to prolong patients chance to survive or improving quality of life are attainable. There are a least 3 important applications for cell free tumor DNA detection. First is to provide information of specific mutation at the time of diagnosis or progression, so that a corresponding targeted therapy can be chosen as the best treatment of choice. Second is to use quantitative amount of specific mutation found in plasma cell free DNA to follow patient’s tumor. The presumption is the amount of specific mutation may represent the tumor load of the treated patients. The 3rd possible application is to use the novel mutations detected in the plasma at the time of diagnosis or during treatment follow up to select a theoretical best combination for patients. There are ample of analysis reported in the literature for the first and second possible applications of plasma cell free DNA. The evidence for the 3rd possible application is accumulating. However, there is no emphasis of using this vast information gathered in the plasma to guide the right timing to switch regimen. In addition, there has been a trend to continue original treatment in slow progressing patients beyond imaging progression, or to treat oligo-progressing sites with local irradiation or surgery in order to keep the original treatment. These approaches certainly will further complicate the rationale decision of right timing to change regimen. Thus, a few options exist for patients who are receiving targeted therapies. One is to use conventional ways of RECIST progression by imaging studies to change the treatment to further lines. The treatment outcome may be more predictable, because most of the clinical trials follow this dogma. A second choice is to treat patients beyond progression by RECIST and follow physicians judgment to switch the regimen. Several recent clinical trials use this criteria to change regimen and collect the treatment time as duration-of-treatment, or time-to-treatment-failure. The 3rd possible timing is to switch regimen according to plasma DNA alterations, for example, emergence of activating mutation when plasma tumor DNA was previously eradicated by current targeted therapy; add another targeted therapy when a new parallel pathways amplification is predicted from the plasma sample analysis. This strategy lack the support of prospective clinical trial data and was purely based on direct scientific deduction or instinct and a few case reports. Therefore, studies specifically designed to address the switch timing is very important. Unfortunately, the effort devoted to this area is lacking.

P2.09-34 Next-Generation Sequencing Implementation in Non-Small Cell Lung Cancer Molecular Diagnosis

P2.09-34 Next-Generation Sequencing Implementation in Non-Small Cell Lung Cancer Molecular Diagnosis

Profiling of circulating tumor DNA in plasma of non-small cell lung cancer patients, monitoring of epidermal growth factor receptor p.T790M mutated allelic fraction using beads, emulsion, amplification, and magnetics companion assay and evaluation in future application in mimicking circulating tumor cells.

Cell‐free plasma DNA (cfDNA) and mimicking circulating tumor cells (mCTCs) have demonstrated tremendous potential for molecular diagnosis of cancer and have been rapidly implemented in specific settings. However, widespread clinical adoption still faces some obstacles. The purpose was to compare the performance of a BEAMing (beads, emulsion, amplification, and magnetics) assay (OncoBEAM™‐epidermal growth factor receptor [EGFR] [Sysmex Inostics]) and a next‐generation sequencing assay (NGS; 56G Oncology panel kit, Swift Bioscience) to detect the p.T790M EGFR mutation in cfDNA of non‐small cell lung cancer (NSCLC) patients. CfDNA samples (n = 183) were collected within our hospital from patients having a known EGFR sensitizing mutation, and presenting disease progression while under first‐line therapy. EGFR mutations were detected using NGS in 42.1% of samples during progression in cfDNA. Testing using the OncoBEAM™‐EGFR assay enabled detection of the p.T790M EGFR mutation in 40/183 NSCLC patients (21.8%) versus 20/183 (10.9%), using the NGS assay. Samples that were only positive with the OncoBEAM™‐EGFR assay had lower mutant allelic fractions (Mean = 0.1304%; SD ± 0.1463%). In addition, we investigated the detection of p.T790M in mCTCs using H1975 cells. These cells spiked into whole blood were enriched using the ClearCellFX1 microfluidic device. Using the OncoBEAM™‐EGFR assay, p.T790M was detected in as few as 1.33 tumoral cells/mL. Overall, these findings highlight the value of using the OncoBEAM™‐EGFR to optimize detection of the p.T790M mutation, as well as the complementary clinical value that each of the mutation detection assay offers: NGS enabled the detection of mutations in other oncogenes that may be relevant to secondary resistance mechanisms, whereas the OncoBEAM™‐EGFR assay achieved higher sensitivity for detection of clinically actionable mutations.