Tumor DNA In Blood Research Articles

Longitudinal Tumor-informed Circulating Tumor DNA Status Predicts Disease Upstaging and Poor Prognosis for Patients Undergoing Radical Cystectomy

Background and objectiveDecision-making on the use of neoadjuvant and adjuvant treatment for patients with bladder cancer undergoing radical cystectomy (RC) currently depends on assessment of clinical and pathological features, which lack sensitivity. Circulating tumor DNA (ctDNA) has emerged as a possible novel prognostic biomarker in the field. Our aim was to assess whether ctDNA status before RC is predictive of pathological and oncological outcomes. We also evaluated the dynamic changes in ctDNA status after RC in relation to recurrence-free survival (RFS). MethodsWe analyzed data for patients who underwent RC during 2021–2023 for whom prospective tumor-informed ctDNA analyses were conducted before and after RC. RFS was evaluated using the Kaplan-Meier method. Predictors for disease recurrence were assessed using Cox proportional-hazards models. Pathological outcomes associated with detectable ctDNA before RC were assessed in univariable and multivariable regression analyses. Key findings and limitationsWe included 112 patients in the analysis. Median follow-up was 8 mo (interquartile range 4–13). ctDNA was detected before RC in 59 patients (53%) and was associated with poor RFS (log-rank p < 0.0001). Detectable ctDNA before RC was associated with poor outcomes regardless of clinical stage (<cT2 vs ≥cT2) and receipt of neoadjuvant therapy. Multivariable analyses revealed that detectable ctDNA before RC was associated with a higher risk of nodal disease (odds ratio 5.4, 95% confidence interval [CI] 1.9–18.2; p = 0.003) and locally advanced disease (odds ratio 3.6, 95% CI 1.5–9; p = 0.005). Cox regression analyses showed that detectable ctDNA before RC (hazard ratio 4.5, 95% CI 1–19; p = 0.04) and detectable ctDNA at the minimal residual disease window (hazard ratio 9.9, 95% CI 2.6–37; p < 0.001) were predictive of disease recurrence. Conclusions and clinical implicationsDetectable ctDNA before definitive therapy with RC is predictive of nodal involvement, locally advanced disease, and disease recurrence in patients with bladder cancer. ctDNA status holds promise for improving clinical staging and augmenting current decision-making tools. Patient summaryWe found that for patients with bladder cancer undergoing radical cystectomy, a test to show the presence of tumor DNA in blood before surgery was able to predict the risk of disease relapse and adverse pathology. Use of this assay could help in decision-making by clinicians and patients for optimal personalized treatment of this disease.

Methylated Circulating Tumor DNA in Blood as a Tool for Diagnosing Lung Cancer: A Systematic Review and Meta-Analysis.

Lung cancer is the leading cause of cancer-related deaths, and early detection is crucial for improving patient outcomes. Current screening methods using computed tomography have limitations, prompting interest in non-invasive diagnostic tools such as methylated circulating tumor DNA (ctDNA). The PRISMA guidelines for systematic reviews were followed. The electronic databases MEDLINE, Embase, Web of Science, and Cochrane Library were systematically searched for articles. The search string contained three main topics: Lung cancer, blood, and methylated ctDNA. The extraction of data and quality assessment were carried out independently by the reviewers. In total, 33 studies were eligible for inclusion in this systematic review and meta-analysis. The most frequently studied genes were SHOX2, RASSF1A, and APC. The sensitivity and specificity of methylated ctDNA varied across studies, with a summary sensitivity estimate of 46.9% and a summary specificity estimate of 92.9%. The area under the hierarchical summary receiver operating characteristics curve was 0.81. The included studies were generally of acceptable quality, although they lacked information in certain areas. The risk of publication bias was not significant. Based on the findings, methylated ctDNA in blood shows potential as a rule-in tool for lung cancer diagnosis but requires further research, possibly in combination with other biomarkers.

A phase 1/2a, multicenter, open-label study of CNA3103 (LGR5-targeted, autologous CAR-T cells) in patients with metastatic colorectal cancer (mCRC).

TPS3632 Background: Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a cancer stem cell (CSC) marker that is abundant in solid tumors, highly expressed in colorectal cancer and involved in tumor initiation, growth, and metastasis. Its expression is linked to poor survival and poor patient response to therapy in mCRC, and presents an attractive opportunity for selectively targeting a highly specific, functional CSC receptor in this disease. CNA3103 is a first-in-class cell product consisting of autologous T cells transduced to express a CAR to target LGR5. It contains the antigen-biding domain of BNC101, a humanized monoclonal LGR5 antibody previously tested in Phase 1 in mCRC subjects and demonstrated to be safe and well tolerated with no off-target binding activity. Methods: This is a first in human, multicenter, open label, Phase 1/2a dose escalation and expansion study to determine the safety of and overall best response to CNA3103 in subjects with mCRC. Up to 44 subjects (24 in Phase 1 and 20 in Phase 2a) will be enrolled. The trial follows a BOIN design with a minimum of 3 consecutively enrolled subjects per cohort, and a 28-day DLT evaluation period. Four cell dose levels (5 × 107, 1.5 × 108, 4.5 × 108, 1.5 × 109) of CNA3103 will be evaluated. A Safety Monitoring Committee (SMC) will oversee the conduct of the study. The pharmacokinetic (PK) profile of CNA3103 in blood will be assessed. Exploratory objectives include evaluation of LGR5+ cells and CNA3103 in tumor biopsies, immune cellular profile, cytokine levels and circulating tumor DNA in blood, and immunogenicity of CNA3103. Main inclusion criteria: mCRC subjects positive for LGR5 expression and failing prior treatment with 5-FU, oxaliplatin and irinotecan-based regimens for metastatic disease; &gt;1 radiologically measurable lesion per RECISTv1.1; &gt;1 biopsiable lesion at baseline; ECOG 0 or 1; serum albumin &gt;3 g/dL; and normal organ and marrow function. Main exclusion criteria: BRAF-mutated disease, clinical ascites/ pleural effusions; active autoimmune/ connective tissue disease; history of inflammatory bowel disease or active peptic ulcer disease. The trial is sponsored by Carina Biotech. Phase 1 enrolment is limited to Australian sites. Phase 2a enrolment will take place in Australia and the US. For information, contact Lina Jablonskis lina@carinabiotech.com or +61 437 891 563.

Circulating tumor DNA in individuals with stomach adenocarcinoma (STAD) to detect MMR-related gene alterations.

e16083 Background: Stomach adenocarcinoma (STAD) ranks as the second incidence rate in China. The MisMatch Repair (MMR), composed of MLH1, MSH2, MSH3, MSH6, and PMS2, is a critical mechanism for repairing DNA error. In case of microsatellite instability (MSI), the MMR system is capable of repairing the mismatches. MSI-high (MSI-H) is created as a result of deficient mismatch repair (dMMR). A numerous studies have established the clinical importance of both MSI and MMR for the diagnosis, prognosis, and treatment of STAD. Circulating tumor DNA (ctDNA) provides a non-invasive method for the diagnosis and prognosis of cancer. The examination of MMR-related gene mutations in STAD can serve as a means of evaluating the utility of ctDNA in detecting. Methods: MMR-related genes list was collected from relevant literature. The clinical and genetic mutation data of 441 STAD samples were obtained from cBioPortal. Kaplan-Meier survival analysis was used to evaluate the total survival rate (OS) of the published STAD data set. 680 STAD patients with TP53 mutation were screened and analyzed from the HapLab database. Results: An analysis of the TCGA data indicated that 356 patients had wild-type MMR genes and 85 had MMR mutations. MSH3 mutations accounted for 19.27% of the overall mutations and 42.35% of patients with MMR mutations, with a total of 36 patients. In terms of OS, patients with MMR gene mutations had a better prognosis compared to those with wild-type genes (log-rank test, p-value = 0.028). In the single-gene prognostic analysis, patients with MSH3 mutations had better prognosis (p-value 0.0004), while no statistical differences were found in other genes. In the monitoring of 680 HapLab STAD patients using ctDNA, 12.35% (84/680) had MMR mutations in their tumor tissue. After treatment, the proportion of TP53 mutations detected in patients was 11.91% (81/680), while the proportion of MMR mutations was 3.57% (3/84). Conclusions: The results of survival analysis showed a significant association between the mutation status of MMR and STAD. Monogenic MSH3 mutations was highly associated with the better survival, and circulating tumor DNA in blood highlight the importance of detecting MMR mutations in STAD patients. ctDNA provides a non-invasive method for the assessment of prognosis in STAD patients through the analysis of MMR gene mutations.

Integrative Oncology's 30-Year Anniversary: What Have We Achieved? A North American Naturopathic Oncology Perspective.

In 1991 the U.S. Congress mandated that the National Institutes of Health (NIH) form the Office of Alternative Medicine to study alternative medical therapies, especially in oncology care. Shortly after, the National Cancer Institute (NCI) created its own division of complementary and alternative medicine (Office of Complementary and Alternative Medicine). At the genesis of the field 30 years ago, what were we hoping to see accomplished by now? In this article we take a look back at milestones, shortfalls and future directions. Exciting opportunities exist to direct our established subspeciality's future directions and we have made valuable advances the field of integrative oncology over the last 30 years: 1, IV high dose ascorbate has clinical research-based applications when used concurrently with some chemotherapeutic agents. 2. Whole body, extracorporeal and locoregional hyperthermia are being applied in treating solid tumors, including brain tumors. 3. PDL-1 tumor microenvironment testing and PDL-1 inhibitor immunotherapies have surprisingly excellent outcomes in a subgroup of cancer patients. 4. Tumor DNA sequencing (resected tumor and circulating tumor DNA in blood) has led to personalized precision targeted treatments. 5. Glucose metabolism's role in cancer progression is better understood and better therapies are available (e.g., intermittent fasting, metformin). 6. Medical cannabis has a larger role in treating chemotherapy-related side effects and shows promise for anti-proliferative effects. 8. Greater understanding has been gained of the interdependence and mutual regulation of processes in psychoneuroendocrinoimmunology (PNEI). The burgeoning field of PNEI has exponentially expanded the discussion of tumorigenesis, apoptosis, and introduced to the field the investigation of more holistic approaches to immune regulation and cancer care. 8. Psychedelic-assisted psychotherapy is gaining traction especially for cancer patients facing demoralization, existential and spiritual distress, anxiety, depression and trauma related to the diagnosis and treatment of their cancer. 9. Spiritual health of cancer patients is more commonly addressed and measurable with an NIH validated scale. 10. Mind-Body therapies are efficacious for reducing cancer-related distress and are included in many cancer care programs.

Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer

Early detection of cancer will improve survival rates. The blood biomarker 5-hydroxymethylcytosine has been shown to discriminate cancer. In a large covariate-controlled study of over two thousand individual blood samples, we created, tested and explored the properties of a 5-hydroxymethylcytosine-based classifier to detect colorectal cancer (CRC). In an independent validation sample set, the classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (95% CI [87, 93]). Sensitivity was 55% at 95% specificity. Performance was similar for early stage 1 (AUC 89%; 95% CI [83, 94]) and late stage 4 CRC (AUC 94%; 95% CI [89, 98]). The classifier could detect CRC even when the proportion of tumor DNA in blood was undetectable by other methods. Expanding the classifier to include information about cell-free DNA fragment size and abundance across the genome led to gains in sensitivity (63% at 95% specificity), with similar overall performance (AUC 91%; 95% CI [89, 94]). We confirm that 5-hydroxymethylcytosine can be used to detect CRC, even in early-stage disease. Therefore, the inclusion of 5-hydroxymethylcytosine in multianalyte testing could improve sensitivity for the detection of early-stage cancer.

C. elegans as a Powerful Tool for Cancer Screening.

Regular cancer screening is critical for early cancer detection. Cancer screening tends to be burdensome, invasive, and expensive, especially for a comprehensive multi-organ check. Improving the rate and effectiveness of routine cancer screenings remain a challenge in health care. Multi-cancer early detection (MCED) is an exciting concept and a potentially effective solution for addressing current issues with routine cancer screening. In recent years, several technologies have matured for MCED, such as identifying cell-free tumor DNA in blood or using organisms such as Caenorhabditis elegans as a tool for early cancer detection. In Japan, N-NOSE is a commercially available multi-cancer detection test based on the chemotaxis of C. elegans using a urine sample showing 87.5% sensitivity and 90.2% specificity. In this review, we focus on using C. elegans as a powerful biosensor for universal cancer screening. We review N-NOSE clinical research results, spotlighting it as an effective primary cancer screening test.

385P The prognostic value of circulating in blood tumor DNA after surgery in I-III stages colorectal cancer

Adjuvant chemotherapy is recommended for all patients with stage III colorectal cancer (CRC) and stage II with unfavorable prognosis factors. To optimize the administration of adjuvant chemotherapy, it is necessary to study markers of high risk of progression. According to the literature data, the prognostic role of circulating tumor DNA (ctDNA) after surgery is shown. The aim of our research was to study the prognostic role of the ctDNA in patients with stage I-III CRC.

Clinical Validity of Tumor-Informed Circulating Tumor DNA Analysis in Patients Undergoing Surgery of Colorectal Metastases.

Accurate biomarkers to monitor tumor load and response in metastatic colorectal cancer patients undergoing surgery could optimize treatment regimens. This study explores the clinical validity of tumor-informed quantification of circulating tumor DNA in blood using ultra-deep sequencing. Resection specimens from 53 colorectal cancer patients were analyzed for tumor-specific mutations in 15 genes. These mutations were used to measure the presence of circulating tumor DNA in preoperatively collected plasma samples using hybrid capture-based sequencing. Additional postoperative measurements were performed one week after surgery in sixteen patients. The study was conducted at the Radboud university medical center. A total of 53 colorectal cancer patients undergoing surgery of metastases were included. The detection of circulating tumor DNA. At least one tumor-specific mutation was detected in all tumor samples. In preoperative plasma samples circulating tumor DNA was detected in 88% (37/42) of systemic treatment-naïve patients and in 55% (6/11) of patients who received preoperative chemotherapy. More specifically, in 0% (0/3) of cases with a subtotal or partial pathological response and in 75% (6/8) of cases without a pathological response in the resection specimen (p = 0.06). In postoperative plasma samples circulating tumor DNA was detected in 80% (4/5) of patients with an incomplete resection and in 0% (0/11) of those with a complete resection (p = 0.003). The study was limited by the heterogeneity of the cohort and the small number of postoperative plasma samples. These data indicate that tumor-informed circulating tumor DNA detection in plasma of patients undergoing surgery for metastatic colorectal cancer is feasible and may have clinical value in response monitoring and predicting residual disease. Prospective studies are needed to establish the clinical utility of circulating tumor DNA analysis to guide treatment decisions in these patients. See Video Abstract at http://links.lww.com/DCR/B990.

The prognostic value of circulating in blood tumor DNA as a marker of minimal residual disease in stage I–III colorectal cancer

Introduction. Studies on non-metastatic colorectal cancer have demonstrated the prognostic role of circulating tumor (ctDNA) after surgery, and the ability to identify patients with the greatest risk of progression. This makes it possible in the future to personalize neoadjuvant and adjuvant treatment.The study objective – to evaluate the correlation of the ctDNA status before and after surgery with a clinical outcome in patients with stage I–III colorectal cancer.Materials and methods. The study included data from patients with morphologically verified colorectal cancer with stage I–III who were treated at the N. N. Blokhin National Oncology Research Center in the period from 2016 to 2021. Blood samples were collected before and after surgical treatment (on the 7–10th day after surgery). The minimum permissible concentration at which ctDNA in a plasma sample was considered positive was 0.4 copies of mutant DNA in 1 mcL of plasma. The main criterion of effectiveness was disease-free survival (DFS). The presence of cDNA before and after surgery was a negative prognostic factor for progression in stage I–III of CRC. Patients with positive cDNA after surgery had worse DFS results despite adjuvant chemotherapy. Patients with stage II CRC with negative ctDNA, regardless of adjuvant CT after surgery, did not have disease progression in 100 % of cases.Results. The study included 146 patients with stage I–III colorectal cancer. Progression was detected in 34 patients. The median follow-up time was 22 months (0–66 months). Data on progression were known in 119 patients. Positive cDNA data were detected before surgery in 55 of 120 patients (45 %), after surgery in 46 of 119 (38.6 %). In the group with positive cDNA before surgery, the median DFS was 35 months (95 % confidence interval (CI) 24,0–45.9), in the group with negative cDNA before surgery, the median DFS was not achieved (hazard ratio (HR) 4.6; 95 % CI 2.0–10.4), 1‑year DFS in the cDNA positive group was 62 % versus 100 % in the cDNA negative group (p &lt;0.001). In the group with positive cDNA after surgery, the median DFS was 20 months (95 % CI 8,1–31,9), in the group with negative cDNA was not achieved (HR 27,7; 95 % CI 6,6–116,6; p &lt;0,001). Patients with positive cDNA after surgery had worse DFS scores despite adjuvant chemotherapy. Patients with stage II CRC without ctDNA after surgery in 100 % did not have disease progression regardless of adjuvant CT.Conclusion. The presence of cDNA before and after surgery was a negative prognostic factor of progression after surgical treatment at stage I–III. The high negative prognostic value of cDNA makes it possible to select patients with stage II who do not need adjuvant chemotherapy.

Liquid Biopsy in Breast Cancer: Circulating Tumor Cells and Circulating Tumor DNA.

Cancer is associated with gene mutations, and the analysis of tumor-associated mutations is increasingly used for diagnostic, prognostic, and treatment purposes. These molecular landscapes of solid tumors are currently obtained from surgical or biopsy specimens. However, during cancer progression and treatment, selective pressures lead to additional genetic changes as tumors acquire drug resistance. Tissue sampling cannot be performed routinely owing to its invasive nature and a single biopsy only provides a limited snapshot of a tumor, which may fail to reflect spatial and temporal heterogeneity. This dilemma may be solved by analyzing cancer cells or cancer cell-derived DNA from blood samples, called liquid biopsy. Liquid biopsy is one of the most rapidly advancing fields in cancer diagnostics and recent technological advances have enabled the detection and detailed characterization of circulating tumor cells and circulating tumor DNA in blood samples.Liquid biopsy is an exciting area with rapid advances, but we are still at the starting line with many challenges to overcome. In this chapter we will explore how tumor cells and tumor-associated mutations detected in the blood can be used in the clinic. This will include detection of cancer, prediction of prognosis, monitoring systemic therapies, and stratification of patients for therapeutic targets or resistance mechanisms.

BIOM-62 SENSITIVE DETECTION AND DISCRIMINATION OF INTRACRANIAL TUMORS BY BLOOD

Abstract BACKGROUND The diagnosis of intracranial tumors relies on tissue specimens obtained by invasive surgery. Non-invasive diagnostic approaches, particularly for patients with brain tumours, provide an opportunity to avoid surgery and mitigate unnecessary risk to patients. We reasoned that DNA methylation profiles of circulating tumor DNA in blood can be used as a clinically useful biomarker for patients with brain tumors, given the specificity of DNA methylation profiles for cell-of-origin. METHODS We generated methylation profiles on the plasma of 608 patients with cancer (219 intracranial, 388 extracranial) and 60 healthy controls using a cell-free methylated DNA immunoprecipitation combined with deep sequencing (cfMeDIP-seq) approach. Using machine-learning approaches we generated and evaluated models to distinguish brain tumors from extracranial cancers that may metastasize to the brain, as well as additional models to discriminate common brain tumors included in the differential diagnosis of solitary extra-axial and intra-axial tumors. RESULTS We observed high sensitivity and discriminative capacity for our models to distinguish gliomas from other cancerous and healthy patients (AUC=0.99, 95%CI 0.96–1), with similar performance in IDH mutant and wildtype gliomas as well as in lower- and high-grade gliomas. Excluding non-malignant contributors to plasma methylation did not change model performance (AUC=0.982, 95%CI 0.93–1). Models generated to discriminate intracranial tumors from each other also demonstrated high accuracy for common extra-axial tumors (AUCmeningioma=0.89, 95%CI 0.80–0.97; AUChemangiopericytoma=0.95, 95%CI 0.73–1) as well as intra-axial tumors ranging from low-grade indolent glial-neuronal tumors (AUC 0.93, 95%CI 0.80 – 1) to diffuse intra-axial gliomas with distinct molecular composition (AUCIDH-mutant glioma = 0.82, 95%CI 0.66 -0.98; AUCIDH-wildtype-glioma = 0.71, 95%CI 0.53 – 0.9). Plasma cfMeDIP-seq signals originated from corresponding tumor tissue DNA methylation signals (r=0.37, p&amp;lt; 2.2e-16). CONCLUSIONS These results demonstrate the potential for cfMeDIP-seq profiles to not only detect circulating tumor DNA, but to accurately discriminate common intracranial tumors that share cell-of-origin lineages.

Efficient mutation screening for cervical cancers from circulating tumor DNA in blood

BackgroundEarly diagnosis and continuous monitoring are necessary for an efficient management of cervical cancers (CC). Liquid biopsy, such as detecting circulating tumor DNA (ctDNA) from blood, is a simple, non-invasive method for testing and monitoring cancer markers. However, tumor-specific alterations in ctDNA have not been extensively investigated or compared to other circulating biomarkers in the diagnosis and monitoring of the CC. Therfore, Next-generation sequencing (NGS) analysis with blood samples can be a new approach for highly accurate diagnosis and monitoring of the CC.MethodUsing a bioinformatics approach, we designed a panel of 24 genes associated with CC to detect and characterize patterns of somatic single-nucleotide variations, indels, and copy number variations. Our NGS CC panel covers most of the genes in The Cancer Genome Atlas (TCGA) as well as additional cancer driver and tumor suppressor genes. We profiled the variants in ctDNA from 24 CC patients who were being treated with systemic chemotherapy and local radiotherapy at the Jeonbuk National University Hospital, Korea.ResultEighteen out of 24 genes in our NGS CC panel had mutations across the 24 CC patients, including somatic alterations of mutated genes (ZFHX3–83%, KMT2C-79%, KMT2D-79%, NSD1–67%, ATM-38% and RNF213–27%). We demonstrated that the RNF213 mutation could be used potentially used as a monitoring marker for response to chemo- and radiotherapy.ConclusionWe developed our NGS CC panel and demostrated that our NGS panel can be useful for the diagnosis and monitoring of the CC, since the panel detected the common somatic variations in CC patients and we observed how these genetic variations change according to the treatment pattern of the patient.

Abstract P5-01-24: DNA palindromes as novel genomic targets for unbiased, genome-wide, and rapid pan-cancer screening

Abstract Cancer continues to have a significant medical, economic, and psychological burden on patients worldwide. One strategy for reducing this impact involves the early detection of cancer biomarkers. Structural chromosomal aberrations called DNA palindromes have immense potential to be used as early markers in tumorigenesis. Palindromic sequences in the genome are structural genetic phenomena where a sequence of nucleotides is identical to its reverse compliment. Various independent studies have identified this distinctive pattern of genome instability in a wide range of cancers including breast, pancreatic, and ovarian cancers. We have shown previously that DNA palindromes underlie the amplification of ERBB2 in breast cancer. This suggests that DNA palindromes are the candidate breast cancer biomarker. A convenient and sensitive approach for identifying palindromes in breast tumor DNA could be a promising diagnostic test. Palindrome detection from tumor DNA in blood (liquid biopsy) could circumvent the need for invasive biopsies. Here, we develop an unbiased genome-wide technique for detecting DNA palindromes as a genomic target for breast cancer screening in the clinic. Using genome-wide analysis of palindrome formation (GAPF), we are able to enrich for DNA palindromes from samples of genomic DNA isolated from cells and tumor tissues. Following a denaturation step, DNA palindromes naturally form double-stranded DNA with itself because these sequences are physically tethered together and can form energetically favorable base pairs. Non-palindromic DNA remains single-stranded and subsequently removed by single-strand specific nucleases thereby dramatically improving the signal-to-noise ratio. This approach confers advantages over other methods because it simultaneously (1) amplifies target signal and (2) reduces background noise without employing sub-sampling techniques. We first used a cancer cell line Colo320DM containing a known palindromic junction and normal fibroblasts IMR90. By integrating GAPF with next generation sequencing (GAPF-Seq), we mapped sequenced reads to a reference genome and developed an algorithm to demarcate DNA palindromes throughout the genome. We are able to identify the known DNA palindrome in Colo320DM in a 10-fold dilution in IMR90 DNA. We then used GAPF-Seq to identify tumor-specific DNA palindromes in matched pairs of tumor and normal breast cancer tissue. From 100 ng of input DNA, we generated a profile of more than 300 tumor-specific DNA palindromes that were not found in the matched normal tissue. To optimize GAPF for use in cancer screening, we expect to detect DNA palindromes in the presence of 100-fold excess of non-palindromic DNA from as little as 10 ng of input DNA. We will then use matched plasma and buffy coat samples to generate whole-genome palindromic profiles from liquid biopsies. The data can also be used for tracing clonal evolution by comparing individual bins in the palindrome profiles of tumor tissue and plasma cell-free DNA from the same patient. Finally, we will optimize GAPF-Seq for minimal computational processing and data storage requirements. Due to the amplification of palindromes and reduction of background signal, GAPF-Seq may be able to identify tumor-specific breast cancer markers from as few as 1 million reads and 250 MB of data, which would be ideal for wide use in screening for breast tumors in the general population and monitoring disease outcomes with multiple blood samplings. We believe GAPF-Seq addresses a need to develop an unbiased, genome-wide, and rapid approach for breast cancer screening. Citation Format: Michael M. Murata, Lila Mouakkad, Dolores Di Vizio, Armando E. Giuliano, Hisashi Tanaka. DNA palindromes as novel genomic targets for unbiased, genome-wide, and rapid pan-cancer screening [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-24.

Intra-Patient Heterogeneity of Circulating Tumor Cells and Circulating Tumor DNA in Blood of Melanoma Patients.

Despite remarkable progress in melanoma therapy, the exceptional heterogeneity of the disease has prevented the development of reliable companion biomarkers for the prediction or monitoring of therapy responses. Here, we show that difficulties in detecting blood-based markers, like circulating tumor cells (CTC), might arise from the translation of the mutational heterogeneity of melanoma cells towards their surface marker expression. We provide a unique method, which enables the molecular characterization of clinically relevant CTC subsets, as well as circulating tumor DNA (ctDNA), from a single blood sample. The study demonstrates the benefit of a combined analysis of ctDNA and CTC counts in melanoma patients, revealing that CTC subsets and ctDNA provide synergistic real-time information on the mutational status, RNA and protein expression of melanoma cells in individual patients, in relation to clinical outcome.

Abstract 1387: Tracking minimal residual disease in post-operative cell-free DNA using MSK-ACCESS

Abstract A noted clinical application for liquid biopsy is as a non-invasive method of detecting and monitoring of minimal residual disease (MRD) in patients with cancer. The low concentration of circulating tumor DNA in blood, especially in early stage cancers, however, complicates the detection of tumor-derived cell-free DNA. To address this challenge, we have developed MSK-ACCESS (Analysis of Circulating cfDNA to Examine Somatic Status), a custom NGS assay covering selected exons from 129 cancer related genes for high-sensitivity detection of somatic mutations from plasma. Using ultra-high depth sequencing, with duplex unique molecular indexing (UMI), unique dual sample barcodes, and background error suppression, MSK-ACCESS is able to detect low-frequency (0.1%) variants with high confidence. The design of MSK-ACCESS leverages our dataset of more than 30,000 tumors profiled by our institutional tumor sequencing assay, MSK-IMPACT, ensuring that the majority of patients harbor multiple mutations that can be tracked in plasma. We have validated MSK-ACCESS using plasma samples collected from 40 healthy individuals and 70 cancer patients harboring a range of somatic mutations in 11 genes. Greater than 95% of mutations at allele fractions &amp;gt;0.1% were empirically detected, and we established the performance characteristics of the assay through intra- and inter-assay reproducibility tests and dilution experiments. We have initiated clinical trials in multiple tumor types to evaluate the benefit of early therapeutic intervention in patients where MSK-ACCESS can detect circulating tumor DNA following surgery. Tumor mutations revealed by MSK-IMPACT in surgically resected specimens will be monitored at regular intervals as evidence of MRD. As a proof of concept, we have applied MSK-ACCESS to monitor variants known from tissue tumor sequencing in pre- and post-surgical cfDNA samples from 9 colon adenocarcinoma patients. All samples were sequenced to an average total depth of approximately 20,000X coverage and subsequently collapsed to consensus sequences exhibiting an average noise level less than 0.0006%. Circulating tumor DNA was detected in 66%(6/9) of the pre-surgical samples. Of these samples, ctDNA was also detected in 50% (3/6) of the post-surgical samples. Overall, this study shows that MSK-ACCESS can be used to successfully detect MRD. Citation Format: Maysun M. Hasan, Juber Patel, Ian Johnson, Fanli Meng, Grittney K. Tam, Xiaohong Jing, Julie L. Yang, A. Rose Brannon, Jayakumaran Gowtham, Dennis P. Stephens, Monica Diosdado, Ryma Benayed, Ahmet Zehir, Chin-Tung Chen, Martin R. Weiser, Dana Tsui, Brian Houck-Loomis, Michael Berger. Tracking minimal residual disease in post-operative cell-free DNA using MSK-ACCESS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1387.

Assessment of Blood Tumor Mutational Burden as a Potential Biomarker for Immunotherapy in Patients With Non–Small Cell Lung Cancer With Use of a Next-Generation Sequencing Cancer Gene Panel

Tumor mutational burden (TMB), as measured by whole-exome sequencing (WES) or a cancer gene panel (CGP), is associated with immunotherapy responses. However, whether TMB estimated by circulating tumor DNA in blood (bTMB) is associated with clinical outcomes of immunotherapy remains to be explored. To explore the optimal gene panel size and algorithm to design a CGP for TMB estimation, evaluate the panel reliability, and further validate the feasibility of bTMB as a clinical actionable biomarker for immunotherapy. In this cohort study, a CGP named NCC-GP150 was designed and virtually validated using The Cancer Genome Atlas database. The correlation between bTMB estimated by NCC-GP150 and tissue TMB (tTMB) measured by WES was evaluated in matched blood and tissue samples from 48 patients with advanced NSCLC. An independent cohort of 50 patients with advanced NSCLC was used to identify the utility of bTMB estimated by NCC-GP150 in distinguishing patients who would benefit from anti-programmed cell death 1 (anti-PD-1) and anti-programmed cell death ligand 1 (anti-PD-L1) therapy. The study was performed from July 19, 2016, to April 20, 2018. Assessment of the Spearman correlation coefficient between bTMB estimated by NCC-GP150 and tTMB calculated by WES. Evaluation of the association of bTMB level with progression-free survival and response to anti-PD-1 and anti-PD-L1 therapy. This study used 2 independent cohorts of patients with NSCLC (cohort 1: 48 patients; mean [SD] age, 60 [13] years; 15 [31.2%] female; cohort 2: 50 patients; mean [SD] age, 58 [8] years; 15 [30.0%] female). A CGP, including 150 genes, demonstrated stable correlations with WES for TMB estimation (median r2 = 0.91; interquartile range, 0.89-0.92), especially when synonymous mutations were included (median r2 = 0.92; interquartile range, 0.91-0.93), whereas TMB estimated by the NCC-GP150 panel found higher correlations with TMB estimated by WES than most of the randomly sampled 150-gene panels. Blood TMB estimated by NCC-GP150 correlated well with the matched tTMB calculated by WES (Spearman correlation = 0.62). In the anti-PD-1 and anti-PD-L1 treatment cohort, a bTMB of 6 or higher was associated with superior progression-free survival (hazard ratio, 0.39; 95% CI, 0.18-0.84; log-rank P = .01) and objective response rates (bTMB ≥6: 39.3%; 95% CI, 23.9%-56.5%; bTMB <6: 9.1%; 95% CI, 1.6%-25.9%; P = .02). The findings suggest that established NCC-GP150 with an optimized gene panel size and algorithm is feasible for bTMB estimation, which may serve as a potential biomarker of clinical benefit in patients with NSCLC treated with anti-PD-1 and anti-PD-L1 agents.

Circulating tumor DNA in blood: Future genomic biomarkers for cancer detection

Cancer is characterized by Darwinian evolution and is a primary cause of mortality and morbidity around the globe. Over the preceding decade, the treatment of cancer has been markedly improved by many targeted therapies, but these treatments have given birth to new challenges and issues. Clonal evolution and tumor heterogeneity present a significant challenge in designing cancer therapies. Fortunately, these restrictions have been overcome by technological advancements allowing us to track both genetic and epigenetic aberrations. Cell-free circulating tumor DNA (ctDNA) analysis, or "liquid biopsy" from a blood sample, provides the opportunity to track the genetic landscape of cancerous lesions. This review focuses on ctDNA analysis as a noninvasive method and versatile biomarker for cancer treatment and technological advancements for ctDNA analysis. This method may able to cope with all the challenges associated with previous cancer therapies and has the potential to monitor minimal residual disease, tumor burden, and therapy response and provide rapid detection of relapse. However, there are many challenges that still need to be addressed. Future prognosis, diagnosis, and analysis of ctDNA require reproducibility and accuracy of results, which are not possible without the validation and optimization of procedures. Integrated digital error suppression has thus far shown promise in the detection of ctDNA in cancer.

Clinical significance of monitoring KRAS in the blood of pancreatic cancer patients.

e23042 Background: KRAS monitoring provides valuable information for early diagnosis and prediction of treatment outcome in colorectal cancer. KRAS mutation is observed in only half of colon cancer patients, whereas it is detected in 90% of pancreatic cancer patients. Therefore, investigating tumor DNA in blood by KRAS monitoring may be even more valuable in pancreatic cancer patients. In this study, we elucidated the clinical significance of KRAS monitoring in pancreatic cancer patients during treatment. Methods: KRAS in tumor tissues was analyzed for mutations by Scorpion ARMS or RASKET in 38 patients with pancreatic tumors. KRAS in blood was analyzed for mutations (G12D, G12V, G12C, G12A, G12S, G12R, G13D, Q61L, and Q61H) using droplet digital polymerase chain reaction in 23 patients who underwent chemotherapy and who had KRAS mutation in their tissues. Results: KRAS mutation in tumor tissues was detected in 27 of 38 patients (71.1%). These 27 patients showed significantly poorer prognosis (24.4 months median overall survival time [MST]) than the six patients without mutation (p = 0.04), who were all alive. Patients with mutation of 12D in tumor tissues showed better prognosis than patients with other types of mutation; thus, mutation of 12D may have a different biological effect to other mutations. Monitoring of KRAS in blood revealed KRAS mutation in 14 of 23 patients (60.9%). These 14 patients exhibited significantly poorer treatment outcomes than patients without mutation. The MST of patients with KRAS mutation in their blood was significantly shorter than that of patients without KRAS mutation (17.7 months vs. 44.3 months, p = 0.03). Conclusions: KRAS mutation in blood could be a valuable predictive and prognostic biomarker in pancreatic cancer patients who undergo chemotherapy. Additionally, assessment of KRAS in tumor tissues may provide information about individual tumor biology.

Statistical modeling for sensitive detection of low-frequency single nucleotide variants.

BackgroundSensitive detection of low-frequency single nucleotide variants carries great significance in many applications. In cancer genetics research, tumor biopsies are a mixture of normal and tumor cells from various subpopulations due to tumor heterogeneity. Thus the frequencies of somatic variants from a subpopulation tend to be low. Liquid biopsies, which monitor circulating tumor DNA in blood to detect metastatic potential, also face the challenge of detecting low-frequency variants due to the small percentage of the circulating tumor DNA in blood. Moreover, in population genetics research, although pooled sequencing of a large number of individuals is cost-effective, pooling dilutes the signals of variants from any individual. Detection of low frequency variants is difficult and can be cofounded by sequencing artifacts. Existing methods are limited in sensitivity and mainly focus on frequencies around 2 % to 5 %; most fail to consider differential sequencing artifacts.ResultsWe aimed to push down the frequency detection limit close to the position specific sequencing error rates by modeling the observed erroneous read counts with respect to genomic sequence contexts. 4 distributions suitable for count data modeling (using generalized linear models) were extensively characterized in terms of their goodness-of-fit as well as the performances on real sequencing data benchmarks, which were specifically designed for testing detection of low-frequency variants; two sequencing technologies with significantly different chemistry mechanisms were used to explore systematic errors. We found the zero-inflated negative binomial distribution generalized linear mode is superior to the other models tested, and the advantage is most evident at 0.5 % to 1 % range. This method is also generalizable to different sequencing technologies. Under standard sequencing protocols and depth given in the testing benchmarks, 95.3 % recall and 79.9 % precision for Ion Proton data, 95.6 % recall and 97.0 % precision for Illumina MiSeq data were achieved for SNVs with frequency > = 1 %, while the detection limit is around 0.5 %.ConclusionsOur method enables sensitive detection of low-frequency single nucleotide variants across different sequencing platforms and will facilitate research and clinical applications such as pooled sequencing, cancer early detection, prognostic assessment, metastatic monitoring, and relapses or acquired resistance identification.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2905-x) contains supplementary material, which is available to authorized users.