Digital PCR Research Articles

Epstein-Barr Virus in the Cerebrospinal Fluid and Blood Compartments of Patients With Multiple Sclerosis and Controls.

Epstein-Barr virus (EBV) infection is a major risk factor of multiple sclerosis (MS). We examined the presence of EBV DNA in the CSF and blood of patients with MS and controls. We analyzed whether EBV DNA is more common in the CSF of patients with MS than in controls and estimated the proportions of EBV-positive B cells in the CSF and blood. CSF supernatants and cells were collected at diagnostic lumbar punctures from 45 patients with MS and 45 HLA-DR15 matched controls with other conditions, all participants were EBV seropositive. Cellular DNA was amplified by Phi polymerase targeting both host and viral DNA, and representative samples were obtained in 28 cases and 28 controls. Nonamplified DNA from CSF cells (14 cases, 14 controls) and blood B cells (10 cases, 10 controls) were analyzed in a subset of participants. Multiple droplet digital PCR (ddPCR) runs were performed per sample to assess the cumulative EBV positivity rate. To detect viral RNA as a sign of activation, RNA sequencing was performed in blood CD4-positive, CD8-positive, and CD19-positive cells from 21 patients with MS and 3 controls. One of the 45 patients with MS and none of the 45 controls were positive for EBV DNA in CSF supernatants (1 mL). CSF cellular DNA was analyzed in 8 independent ddPCRs: EBV DNA was detected at least once in 18 (64%) of the 28 patients with MS and in 15 (54%) of the 28 controls (p = 0.59, Fisher test). The cumulative EBV positivity increased steadily up to 59% in the successive ddPCRs, suggesting that all individuals would have reached EBV positivity in the CSF cells, if more DNA would have been analyzed. The estimated proportion of EBV-positive B cells was >1/10,000 in both the CSF and blood. We did not detect viral RNA, except from endogenous retroviruses, in the blood lymphocyte subpopulations. EBV-DNA is equally detectable in the CSF cells of both patients with MS and controls with ddPCR, and the probabilistic approach indicates that the true positivity rate approaches 100% in EBV-positive individuals. The proportion of EBV-positive B cells seems higher than previously estimated.

Droplet digital PCR: An effective method for monitoring and prognostic evaluation of minimal residual disease in JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative neoplasm requiring haematopoietic stem cell transplantation (HSCT) for potential cure. Relapse poses a significant obstacle to JMML HSCT treatment, as the lack of effective minimal residual disease (MRD)-monitoring methods leads to delayed interventions. This retrospective study utilized the droplet digital PCR (ddPCR) technique, a highly sensitive nucleic acid detection and quantification technique, to monitor MRD in 32 JMML patients. The results demonstrated that ddPCR detected relapse manifestations earlier than traditional methods and uncovered molecular insights into JMML MRD dynamics. The findings emphasized a critical 1- to 3-month window post-HSCT for detecting molecular relapse, with 66.7% (8/12) of relapses occurring within this period. Slow MRD clearance post-HSCT was observed, as 65% (13/20) of non-relapse patients took over 6 months to achieve ddPCR-MRD negativity. Furthermore, bone marrow ddPCR-MRD levels at 1-month post-HSCT proved to be prognostically significant. Relapsed patients exhibited significantly elevated ddPCR-MRD levels at this time point (p = 0.026), with a cut-off of 0.465% effectively stratifying overall survival (p = 0.007), event-free survival (p = 0.035) and cumulative incidence of relapse (p = 0.035). In conclusion, this study underscored ddPCR's superiority in JMML MRD monitoring post-HSCT. It provided valuable insights into JMML MRD dynamics, offering guidance for the effective management of JMML.

Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Novel milestones for early esophageal carcinoma: From bench to bed

Esophageal cancer (EC) is the seventh most common cancer worldwide, and esophageal squamous cell carcinoma (ESCC) accounts for the majority of cases of EC. To effectively diagnose and treat ESCC and improve patient prognosis, timely diagnosis in the initial phase of the illness is necessary. This article offers a detailed summary of the latest advancements and emerging technologies in the timely identification of ECs. Molecular biology and epigenetics approaches involve the use of molecular mechanisms combined with fluorescence quantitative polymerase chain reaction (qPCR), high-throughput sequencing technology (next-generation sequencing), and digital PCR technology to study endogenous or exogenous biomolecular changes in the human body and provide a decision-making basis for the diagnosis, treatment, and prognosis of diseases. The investigation of the microbiome is a swiftly progressing area in human cancer research, and microorganisms with complex functions are potential components of the tumor microenvironment. The intratumoral microbiota was also found to be connected to tumor progression. The application of endoscopy as a crucial technique for the early identification of ESCC has been essential, and with ongoing advancements in technology, endoscopy has continuously improved. With the advancement of artificial intelligence (AI) technology, the utilization of AI in the detection of gastrointestinal tumors has become increasingly prevalent. The implementation of AI can effectively resolve the discrepancies among observers, improve the detection rate, assist in predicting the depth of invasion and differentiation status, guide the pericancerous margins, and aid in a more accurate diagnosis of ESCC.

D-Loop Mutations as Prognostic Markers in Glioblastoma—A Pilot Study

Glioblastoma, a highly aggressive brain tumor, poses significant treatment challenges. A deeper investigation into its molecular complexity is essential for the identification of novel prognostic biomarkers and therapeutic strategies, potentially improving patient outcomes in terms of survival and quality of life. While nuclear DNA mutations have been extensively studied, the role of mitochondrial DNA (mtDNA) mutations, specifically in the D-loop region, remains poorly understood. This prospective case-control study aimed to assess the prognostic significance of the mtDNA D-loop m.16126T>C variant in glioblastoma patients. Immunohistochemistry and droplet digital PCR (ddPCR) were employed for mutation analysis, complemented by statistical analyses and a literature review. The study cohort comprised 22 glioblastoma patients (mean age 59.36 ± 14.17, 12 (54.55%) females), and 25 controls (59.48 ± 13.22, 12 (80%) females). The D-loop m.16126T>C variant was observed in four (18%) of the glioblastoma samples and was associated with shorter median survival (9.5 vs. 18 months; p = 0.016, log-rank test). This study underscores the importance of investigating mtDNA, especially D-loop variants, in glioblastoma, suggesting its potential as a prognostic biomarker and, therefore, its possible therapeutic targets, warranting further exploration.

Effect of a 12-Week Walking Program Monitored by Global Physical Capacity Score (GPCS) on Circulating Cell-Free mtDNA and DNase Activity in Patients with Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) involves low-grade mucosal inflammation. Among the various approaches capable of managing the symptoms, physical activity is still under investigation. Despite its benefits, it promotes oxidative stress and inflammation. Mitochondria impacts gut disorders by releasing damage-associated molecular patterns, such as cell-free mtDNA (cf-mtDNA), which support inflammation. This study evaluated the effects of a 12-week walking program on the cf-mtDNA and DNase in 26 IBS and 17 non-IBS subjects. Pro- and anti-inflammatory cytokines were evaluated by ELISA. Digital droplet PCR was used to quantify cf-mtDNA; DNase activity was assessed using a single radial enzyme diffusion assay. PCR-RFLP was used to genotype DNASE1 rs1053874 SNP. Significantly lower IL-10 levels were found in IBS than in non-IBS individuals. Exercise reduced cf-mtDNA in non-IBS subjects but not in IBS patients. DNase activity did not correlate with the cf-mtDNA levels in IBS patients post-exercise, indicating imbalanced cf-mtDNA clearance. Different rs1053874 SNP frequencies were not found between groups. The study confirms the positive effects of regular moderate-intensity physical activity in healthy subjects and its role in cf-mtDNA release and clearance. Walking alone might not sufficiently reduce subclinical inflammation in IBS, based on imbalanced pro- and anti-inflammatory molecules. Prolonged programs are necessary to investigate their effects on inflammatory markers in IBS.

Bacterial and viral co-infections in aquaculture under climate warming: co-evolutionary implications, diagnosis, and treatment.

Climate change and the associated environmental temperature fluctuations are contributing to increases in the frequency and severity of disease outbreaks in both wild and farmed aquatic species. This has a significant impact on biodiversity and also puts global food production systems, such as aquaculture, at risk. Most infections are the result of complex interactions between multiple pathogens, and understanding these interactions and their co-evolutionary mechanisms is crucial for developing effective diagnosis and control strategies. In this review, we discuss current knowledge on bacteria-bacteria, virus-virus, and bacterial and viral co-infections in aquaculture as well as their co-evolution in the context of global warming. We also propose a framework and different novel methods (e.g. advanced molecular tools such as digital PCR and next-generation sequencing) to (1) precisely identify overlooked co-infections, (2) gain an understanding of the co-infection dynamics and mechanisms by knowing species interactions, and (3) facilitate the development multi-pathogen preventive measures such as polyvalent vaccines. As aquaculture disease outbreaks are forecasted to increase both due to the intensification of practices to meet the protein demand of the increasing global population and as a result of global warming, understanding and treating co-infections in aquatic species has important implications for global food security and the economy.

Low C4A copy numbers and higher HERV gene insertion contributes to increased risk of SLE, with absence of association with disease phenotype and disease activity.

Low copy numbers (CNs) of C4 genes are associated with systemic autoimmune disorders and affects autoantibody diversity and disease subgroups. The primary objective of this study was to characterize diversity of complement (C4) and C4-Human Endogenous Retrovirus (HERV) gene copy numbers in SLE. We also sought to assess the association of C4 and C4-HERV CNs with serum complement levels, autoantibodies, disease phenotypes and activity. Finally, we checked the association of C4 and HERV CNs with specific HLA alleles. Genomic DNA from 70 SLE and 90 healthy controls of south Indian Tamil origin were included. Demographic, clinical and serological data was collected in a predetermined proforma. CNs of C4A and C4B genes and the frequency of insertion of 6.4kb HERV within C4 gene (C4AL, C4BL) was determined using droplet digital polymerase chain reaction (ddPCR). A four digit high resolution HLA genotyping was done using next generation sequencing. In our cohort, the total C4 gene copies ranged from 2 to 6. Compared to controls, presence of two or less copies of C4A gene was associated with SLE risk (p = 0.005; OR = 2.79; 95% CI = 1.29-6.22). Higher frequency of HERV insertion in C4A than in C4B increases such risk (p = 0.000; OR = 12.67; 95% CI = 2.80-115.3). AL-AL-AL-BS genotype was significantly higher in controls than SLE (9%vs1%, p = 0.04; OR = 0.15, 95% CI = 0.00-0.16). Distribution of HLA alleles was not different in SLE compared to controls as well as in SLE subjects with ≤ 2 copies and > 2 copies of C4A, but HLA allele distribution was diverse in subjects with C4B ≤ 2 copies and > 2 copies. Finally, there was no correlation between the C4 and the C4-HERV diversity and complement levels, autoantibodies, disease phenotypes and activity. In conclusion, our data show that, low C4A copy number and higher insertion of HERV-K in C4A increases the risk for SLE. C4 and C4-HERV CNs did not correlate with serum complements, autoantibodies, disease phenotypes and activity in SLE. Further validation in a larger homogenous SLE cohort is needed.

Genome-wide identification and characterisation of hevein precursor genes in Hevea brasiliensis

Hevea brasiliensis is the primary source of natural rubber, which is obtained from its latex and used in the manufacturing of various products. Hevein is a small protein found in the latex, produced by post-translational cleavage of prohevin, which is encoded by the hevein precursor gene. It exhibits antimicrobial and agglutination properties. Several nucleotide sequences that encode the hevein precursor genes were reported, however, the sequences are not characterised well. In the present study, four full-length hevein precursor sequences (Hevein 1–4) were obtained by manually curating the sequences from different databases. All sequences show high homology with the highest identity between Hevein 1 and Hevein 3. In a phylogenetic analysis along with sequences from related plants, all sequences from H. brasiliensis were clustered into a specific clade. All hevein precursor genes were expressed in the latex samples obtained from three clones; RRIM 600, RRIM 3001 and PB 350 of less than one year old and five-year-old RRIM 3001 plants, grown in plant house, as well as field-grown trees of clone PB 350 belonging to three different ages; two years, six years and fifteen years. All hevein precursor genes in the five-year-old RRIM 3001 plants with stunted growth, maintained in the plant house showed alternative splicing. Hevein 3 was expressed with two splice variants, one with intron retention and the other without intron whereas the other genes were expressed with only intron retained variant. Differential expression analysis using nanoplate digital PCR showed that Hevein 2 and Hevein 3 were expressed with no significant difference among the three young H. brasiliensis clones. The expression of Hevein 2 and Hevein 3 among the H. brasiliensis clone PB 350 of different ages grown in field conditions showed significant difference. The present study provides a better understanding on the importance of hevein precursor genes in different physiological responses which will be useful for further research leading to the genetic improvement of H. brasiliensis.

Diversity Analysis of Intestinal Bifidobacteria in the Hohhot Population

(1) Background: Bifidobacterium plays a pivotal role within the gut microbiota, significantly affecting host health through its abundance and composition in the intestine. Factors such as age, gender, and living environment exert considerable influence on the gut microbiota, yet scant attention has been directed towards understanding the specific effects of these factors on the Bifidobacterium population. Therefore, this study focused on 98 adult fecal samples to conduct absolute and relative quantitative analyses of bifidobacteria. (2) Methods: Using droplet digital PCR and the PacBio Sequel II sequencing platform, this study sought to determine the influence of various factors, including living environment, age, and BMI, on the absolute content and biodiversity of intestinal bifidobacteria. (3) Results: Quantitative results indicated that the bifidobacteria content in the intestinal tract ranged from 106 to 109 CFU/g. Notably, the number of bifidobacteria in the intestinal tract of the school population surpassed that of the off-campus population significantly (p = 0.003). Additionally, the group of young people exhibited a significantly higher count of bifidobacteria than the middle-aged and elderly groups (p = 0.041). The normal-weight group displayed a significantly higher bifidobacteria count than the obese group (p = 0.027). Further analysis of the relative abundance of bifidobacteria under different influencing factors revealed that the living environment emerged as the primary factor affecting the intestinal bifidobacteria structure (p = 0.046, R2 = 2.411). Moreover, the diversity of bifidobacteria in the intestinal tract of college students surpassed that in the out-of-school population (p = 0.034). This was characterized by a notable increase in 11 strains, including B. longum, B. bifidum, and B. pseudolongum, in the intestinal tract of college students, forming a more intricate intestinal bifidobacteria interaction network. (4) Conclusions: In summary, this study elucidated the principal factors affecting intestinal bifidobacteria and delineated their characteristics of intestinal bifidobacteria in diverse populations. By enriching the theory surrounding gut microbiota and health, this study provides essential data support for further investigations into the intricate dynamics of the gut microbiota.

Genomic and Pathologic Profiling of Very Well-Differentiated Gastric Adenocarcinoma of Intestinal Type: A Study With Emphasis on Diffuse-Type Transformation.

Very well-differentiated adenocarcinoma of intestinal type is a distinct subtype of gastric cancer characterized by anastomosing glands with a hand-in-hand pattern and low-grade cytologic atypia resembling intestinal metaplasia. This is a slow-growing neoplasm with an indolent clinical course; however, a subset demonstrates transformation into adenocarcinoma with higher-grade histology, typically diffuse-type carcinoma, and behaves aggressively. This study aimed to better characterize the genomic and pathologic features, with a focus on factors associated with diffuse-type transformation. A total of 58 cases with (n=31) and without (n=27) diffuse-type transformation were analyzed for molecular and pathologic features. First, comprehensive deep DNA sequencing was conducted in 18 cases (discovery cohort), followed by a digital droplet polymerase chain reaction of hot spot RHOA mutations in 40 cases (validation cohort). In total, RHOA mutations were the most common alteration (34%), followed by loss of ARID1A (12%), p53 alterations (10%), and CLDN18::ARHGAP26/6 fusions (3.4%). FGFR2 amplification was identified in an advanced case with a p53 alteration. Altered p53 expression was recognized only in higher-grade components and was significantly associated with advanced disease (P=0.0015) and diffuse-type transformation (P=0.026). A mixed mucin phenotype was also strongly correlated with advanced disease (P<0.001) and diffuse-type transformation (P<0.001). Decreased E-cadherin expression was frequently observed (74%) in poorly cohesive components. This study demonstrated that a subset of RHOA-mutant diffuse-type gastric cancers develops through the transformation of very well-differentiated adenocarcinoma of intestinal type. Our observations suggest a mixed mucin phenotype as a risk factor and alterations in p53 and E-cadherin as drivers of diffuse-type transformation.

Identification of a mosaic MTOR variant in purified neuronal DNA in a patient with focal cortical dysplasia using a novel depth electrode harvesting technique.

Recent studies have identified brain somatic variants as a cause of focal epilepsy. These studies relied on resected tissue from epilepsy surgery, which is not available in most patients. The use of trace tissue adherent to depth electrodes used for stereo electroencephalography (EEG) has been proposed as an alternative but is hampered by the low cell quality and contamination by nonbrain cells. Here, we use our improved depth electrode harvesting technique that purifies neuronal nuclei to achieve molecular diagnosis in a patient with focal cortical dysplasia (FCD). Depth electrode tips were collected, pooled by brain region and seizure onset zone, and nuclei were isolated and sorted using fluorescence-activated nuclei sorting (FANS). Somatic DNA was amplified from neuronal and astrocyte nuclei using primary template amplification followed by exome sequencing of neuronal DNA from the affected pool, unaffected pool, and saliva. The identified variant was validated using droplet digital polymerase chain reaction (PCR). An 11-year-old male with drug-resistant genetic-structural epilepsy due to left anterior insula FCD had seizures from age 3 years. Stereo EEG confirmed seizure onset in the left anterior insula. The two anterior insula electrodes were combined as the affected pool and three frontal electrodes as the unaffected pool. FANS isolated 140 neuronal nuclei from the affected and 245 neuronal nuclei from the unaffected pool. A novel somatic missense MTOR variant (p.Leu489Met, CADD score 23.7) was identified in the affected neuronal sample. Droplet digital PCR confirmed a mosaic gradient (variant allele frequency = .78% in affected neuronal sample; variant was absent in all other samples). Our findings confirm that harvesting neuronal DNA from depth electrodes followed by molecular analysis to identify brain somatic variants is feasible. Our novel method represents a significant improvement compared to the previous method by focusing the analysis on high-quality cells of the cell type of interest.

Abstract CT017: FLAURA2: exploratory analysis of baseline (BL) and on-treatment plasma EGFRm dynamics in patients (pts) with EGFRm advanced NSCLC treated with first-line (1L) osimertinib (osi) ± platinum-pemetrexed

Abstract Background In FLAURA2, 1L osi with addition of platinum-pemetrexed chemotherapy (osi + CTx) significantly improved PFS vs osi alone in pts with EGFRm advanced NSCLC. Prior FLAURA analyses showed detected BL plasma EGFRm to be prognostic and early clearance of plasma EGFRm correlated with improved outcomes. We explored correlation of BL plasma EGFRm detection, and its clearance, with PFS in FLAURA2 to determine its potential to identify pts who derive particular benefit from osi + CTx. Methods Treatment-naïve pts with EGFRm (Ex19del or L858R) advanced NSCLC were randomized to osi + CTx (osi 80 mg once daily [QD] + pemetrexed 500 mg/m2 + cisplatin 75 mg/m2 or carboplatin AUC5 every 3 wks [Q3W] for 4 cycles, followed by osi 80 mg QD + pemetrexed 500 mg/m2 Q3W) or osi alone (80 mg QD). BL and wk 3/6 plasma EGFRm were analyzed by ddPCR (Biodesix). Clearance was defined as undetected EGFRm circulating tumor DNA at wk 3/6, where it was detected at BL. Median PFS (mPFS) was investigator-assessed per RECIST 1.1. Results In total, 421/557 pts had an evaluable BL plasma ddPCR result, of who 308 had detected BL plasma EGFRm. Of pts randomized to osi alone, pts with non-detected BL plasma EGFRm had longer mPFS* vs those with detected BL plasma EGFRm. Of pts with evaluable BL and wk 3/6 plasma ddPCR results, plasma EGFRm was detected at wk 3/6 in 19/12% of osi + CTx-treated pts, and 29/11% of osi-treated pts, respectively (Table). Conclusion In this exploratory analysis, detected BL plasma EGFRm was prognostic and appeared predictive of clinical benefit with osi + CTx vs osi alone. Detection of BL plasma EGFRm may identify a subgroup of pts deriving most benefit from the addition of CTx to osi. Although within each treatment arm plasma EGFRm wk3 clearance correlated with improved PFS, the relative benefit of osi + CTx vs osi alone was similar, suggestive that on-treatment EGFRm clearance is only prognostic in this setting. Osimertinib + CTx (evaluable BL ddPCR, n = 212) Osimertinib (evaluable BL ddPCR, n = 209) Osimertinib + CTx vs osimertinib n mPFS (mo) n mPFS (mo) HR (95% CI) Baseline plasma EGFRm Detected 147 24.8 161 13.9* 0.60 (0.45, 0.80) Not detected 65 33.3 48 30.3* 0.93 (0.51, 1.72) EGFRm clearance/non-clearance at 3 weeks Clearance 88 27.6 100 15.2 0.51 (0.35, 0.75) No clearance 21 19.6 40 11.1 0.62 (0.33, 1.14) *Not detected vs detected in the osimertinib arm: mPFS HR 0.51 (95% CI 0.35, 0.76). BL, baseline; CI, confidence interval; CTx, chemotherapy; ddPCR, droplet digital PCR; EGFRm, epidermal growth factor receptor mutation-positive; HR, hazard ratio; mo, months; mPFS, median progression-free survival Citation Format: Pasi A. Jänne, Kunihiko Kobayashi, Jacqulyne Robichaux, Chee Khoon Lee, Shunichi Sugawara, Tsung-Ying Yang, Tae Min Kim, Noriko Yanagitani, Sang-We Kim, Aleksandra Markovets, Preetida Bhetariya, Lynne Poole, Dana Ghiorghiu, Ryan Hartmaier, James Chih-Hsin Yang, David Planchard. FLAURA2: exploratory analysis of baseline (BL) and on-treatment plasma EGFRm dynamics in patients (pts) with EGFRm advanced NSCLC treated with first-line (1L) osimertinib (osi) ± platinum-pemetrexed [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT017.

Abstract LB094: Assessment of minimal residual disease in lung cancer patients treated with osimertinib using liquid biopsy

Abstract Purpose: Drug tolerance has emerged as one of the major mechanisms driving resistance to targeted therapies (TT), but a comprehensive understanding of the dynamics and molecular heterogeneity underlying the adaptive drug response in patients is still lacking. Here, we used iterative liquid biopsies from osimertinib-treated lung cancer patients to monitor and characterize minimal residual disease (MRD) using both circulating tumor DNA (ctDNA) and circulating tumor cells (CTC). Experimental design: LUNG-RESIST (NCT04222335) is a prospective research study enrolling EGFR-mutant adenocarcinoma patients treated by osimertinib frontline. We collected blood samples at baseline, 1 month and every 3 months until progression. CtDNA was used to monitor the initial EGFR mutation (EGFRm) by digital PCR, and to identify new potential biomarkers of resistance by next-generation sequencing (NGS). CTC were enriched and isolated based on their size, absence of leucocyte staining and viability, to perform single-cell transcriptomic and genomic analyses. Results: 40 patients have been enrolled with a median progression-free survival (mPFS) of 14.5 months (95%CI, 8.5-24.5 months). 28/40 (70%) patients had detectable EGFRm in ctDNA at baseline, and decreased ctDNA level could be measured in 25/26 (96%) patients after 1 month of treatment, with a complete clearance in 15/26 (58%) patients. 7/11 (64%) patients without clearance at 1 month relapsed within 9 months. Clinical relapse could be anticipated 3 months ahead based on ctDNA increase for 9/20 (45%) patients. CTC-like cells have been collected at baseline and MRD (between 1 and 3 months) from respectively 31/40 (78%) and 29/40 (73%) patients, and at clinical progression for 12/22 (54%) patients who relapsed, for a total number of 1,510 cells. The number of CTC-like cells decreased after 1 month of treatment in 18/29 (62%) patients, and increased again in 7/12 (58%) patients at the time of clinical relapse. The results of NGS analyses on ctDNA and single-cell transcriptomic and genomic analyses on CTC will be presented at the congress, and should provide a better insight into the dynamics of molecular events occurring throughout the adaptive response in patients. Conclusion: We report the first molecular profiling of both ctDNA and CTC during the adaptive response to osimertinib in lung cancer patients. Monitoring the response and predicting the clinical outcome for EGFR-mutant lung cancer patients undergoing TT treatment can be done using ctDNA level as a biomarker. The isolation of CTC-like cells represents a non-invasive approach to study drug tolerance and ultimately develop new combinatory treatments to prevent relapse. Citation Format: Celia Delahaye, Anne Casanova, Estelle Clermont-Tarenchon, Aurelia Doussine, Juan-Pablo Cerapio, Anaïs Anton, Emma Devienne, Vincent Dongay, Julie Milia, Gilles Favre, Olivier Calvayrac, Anne Pradines, Julien Mazieres. Assessment of minimal residual disease in lung cancer patients treated with osimertinib using liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB094.

Abstract CT002: CTX130 allogeneic CRISPR-Cas9-engineered chimeric antigen receptor (CAR) T cells in patients with advanced clear cell renal cell carcinoma: Long-term follow-up and translational data from the phase 1 COBALT-RCC study

Abstract Background: The mainstay of therapy for clear cell renal cell carcinoma (ccRCC), the most prevalent subtype of kidney cancer, includes checkpoint inhibitors (CPIs) and tyrosine kinase inhibitors (TKIs). However, once patients progress on these drug classes, outcomes are poor. Here, we report durable response and stability observed in a Phase 1 dose-escalation study of CTX130™, a CD70-targeting allogeneic CAR T cell therapy in patients with ccRCC. Methods: In this phase I study, patients were recruited from sites in the United States, Europe and Australia. Key eligibility included advanced ccRCC with prior receipt of at least one CPI and TKI. After receipt of standard lymphodepletion with fludarabine and cyclophosphamide for 3 days (dosed at 30 mg/m2 and 500 mg/m2, respectively), patients received infusion of CTX130. Results: Among the 16 patients enrolled and treated in dose escalation, the median age was 63 (range, 53-77) and all were stage IV at the time of enrollment. Patients had received a median of 3 prior lines of therapy (range, 1-6). Doses ranging from 3 × 107 to 9 × 108 CAR T cells were administered, and no dose-limiting toxicities were encountered. Cytokine release syndrome (CRS) was observed in 8 patients (50%); all events were grade 1 or 2. Infections were observed in 4 patients (25%), all deemed to be unrelated to CTX130. No hemophagocytic lymphohistiocytosis, graft-versus host disease or treatment-related neurotoxicity was observed. As of Oct 9, 2023, 1 patient (6.3%) achieved a complete response (CR) (now extending over 36+ months) and 12 additional patients (75%) achieved stable disease (SD) (1 patient in excess of 18 months), reflecting a disease control rate (CR/partial response/SD) of 81.3%. Immunohistochemical staining of baseline and archival tissues show an initial median CD70 expression level of 100% (range, 1-100%), with no substantial antigenic loss at sequential biopsies performed at day 42. Using digital droplet PCR assays of the CAR construct, we assessed expansion. CTX130 was detected in 20 minutes after infusion, declined to a nadir 2-3 days later, followed by rapid expansion between days 7 and 15. In general, expansion increased at each dose level. Conclusions: Updated results from the COBALT-RCC study reinforce both the encouraging antitumor activity and exceptional safety profile of CTX130. The durable CR, now in excess of 3 years, reflects the first and longest seen with allogeneic CAR T cell therapy in refractory solid tumors. A study of CTX131™ (building on CTX130 with CRISPR edits to enhance potency and persistence) is ongoing. Citation Format: Sumanta Kumar Pal, Ben Tran, John B. Haanen, Michael Hurwitz, Adrian Sacher, Neeraj Agarwal, Nizar Tannir, Elizabeth Budde, Simon Harrison, Sebastian Klobuch, Sagar S. Patel, Mary-Lee Dequeant, Qiuling Ally He, Alissa Keegan, Henia Dar, Anna Ma, PK Morrow, Samer A. Srour. CTX130 allogeneic CRISPR-Cas9-engineered chimeric antigen receptor (CAR) T cells in patients with advanced clear cell renal cell carcinoma: Long-term follow-up and translational data from the phase 1 COBALT-RCC study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT002.

Development of a multiplex droplet digital PCR method for detection and monitoring of Mycobacterium tuberculosis and drug-resistant tuberculosis

BackgroundThe prevalence of multidrug-resistant tuberculosis (MDR-TB) among Korean tuberculosis patients is about 4.1%, which is higher than the OECD average of 2.6%. Inadequate drug use and poor patient compliance increase MDR-TB prevalence through selective pressure. Therefore, prompt detection of drug resistance in tuberculosis patients at the time of diagnosis and quantitative monitoring of these resistant strains during treatment are crucial.MethodsA multiplex droplet digital PCR (ddPCR) assay was developed and assessed using DNA material of nine Mycobacterium tuberculosis strains with known mutation status that were purchased from the Korean National Tuberculosis Association. We collected a total of 18 MDR-TB residual samples referred for PCR analysis. Total DNA was extracted from the samples and subjected to the quadruplex ddPCR assay. Their results were compared to those of known resistance phenotypes.ResultsThe analytical sensitivity and specificity of the multiplex ddPCR assay for detecting INH, RIF, EMB, FQ, and SM resistance-causing mutations ranged from 71.43 to 100% and 94.12–100%, respectively. Follow-up sample results showed that the quadruplex ddPCR assay was sensitive enough to detect IS6110 and other mutations even after onset of treatment.ConclusionsWe developed a sensitive and accurate multiplex ddPCR assay that can detect the presence of tuberculosis quantitatively and resistance-conveying mutations concurrently. This tool could aid clinicians in the diagnosis and treatment monitoring of tuberculosis.

Abstract LB099: Identifying actionable genomic alterations in non-small cell lung cancer through the analysis of extracellular vesicles in plasma

Abstract Lung cancer is the second most common cancer worldwide, accounting for 2.2 million new cases and 1.8 million deaths in 2020. Personalized targeted therapy based on actionable molecular markers has completely transformed the therapeutic landscape in NSCLC, which has improved considerably the overall survival. However, ~50% of NSCLC patients with tissue biopsy inaccessible or inadequate, limiting biomarker testing and access to targeted therapies. The availability of a wide range of highly effective targeted therapy options for NSCLC justifies the significance of developing non-invasive and cost-effective detection assays for guiding targeted therapy selection in NSCLC.Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles of sub-micrometer size that are secreted by virtually all cell types. They contain hundreds of different proteins, thousands of intact RNA species, and double-stranded DNA (dsDNA) fragments that sample the entire human genome. Recent findings further corroborate the potential role of EVs in early cancer screening, cancer diagnosis, treatment selection and monitoring. In this study, we used lipid nanoprobe (LNP) for EV isolation from plasma and droplet digital PCR assay for detecting actionable genomic alternation to aid targeted therapy selection in NSCLC. Specifically, we extracted EV associated DNA/RNA from plasma first, and then we used the individual ddPCR assays for detecting KRAS mutations, EGFR mutations, and ALK fusions from isolated EVs associated nucleic acids, respectively. We identified KRAS mutations G12C/G12S/G13C/G12A/Q61H, EGFR L858R mutation and exon 19 deletion, and ALK-EML4 gene fusion variant 3 from 12 NSCLC patients that matched the mutation status obtained from patient tissue specimens. Only in 1 out 13 patients, EV mutation status did not match the mutation status from the tissue specimen. This data indicated that clinical actionable gene mutations are present in EVs associated nucleic acids isolated from plasma samples of NSCLC patients. We anticipate that this novel non-invasive EVs-based actionable genomic alterations detection test would efficiently assist physicians/oncologists in selecting optimal targeted therapies for cancer treatment, as recommended by National Comprehensive Cancer Network (NCCN) guidelines for NSCLC. Citation Format: Hongzhang He, Jadranka Milosevic, Siyang Zheng. Identifying actionable genomic alterations in non-small cell lung cancer through the analysis of extracellular vesicles in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB099.

Abstract LB106: Ultra-short ctDNA: A new paradigm for ctDNA liquid biopsy

Abstract Mononucleosomal ctDNA (mnctDNA, 157-bp, double-stranded) spearheads liquid biopsy (LB) for cancer detection, treatment and clinical care. The bottleneck of mnctDNA LB are low copy number of ctDNA and limit of detection of platform technologies. Recent discovery of ultra-short ctDNA (usctDNA, 52-54 nt, single-stranded) that harbors somatic mutations prompted us to explore the quantification and stoichiometry of usctDNA to mnctDNA in plasma and saliva of NSCLC patients. Paired plasma and saliva from 20 advanced NSCLC patients with tissue genotyped positive for L858R were processed for cfDNA extraction and quantification with Qubit and Tape Station. Digital PCR assays targeting L858R somatic mutation to generate amplicons of 57-bp (usctDNA) and 100-bp (mnctDNA) were performed by UCLA and independently by NIST. Results in Table 1 revealed that three findings of usctDNA that will have potential to impact ctDNA liquid biopsy. First, the somatic mutation (L858R) is present in usctDNA. Second, The stoichiometric ratio of usctDNA to mnctDNA is similar in plasma and saliva at 1.6X. For the wild-type counterpart, the ratio is 1.1X for plasma and 0.6X for saliva. Third, the copy number of usctDNA and mnctDNA in saliva to plasma is 7.8X and 7.7X. Conclusion: Detection of tumor-associated/specific EGFR L858R somatic mutations in uscfDNA is translationally impactful, adding 1.6X of the ctDNA targets for molecular detection, effectively cumulating 2.6X of total ctDNA targets, permitting earlier cancer detection, treatment monitoring and therapy prognostication. The finding that saliva harbors 7X of the us- and mnctDNA further raises the prospect of advancing saliva ctDNA liquid biopsy for NSCLC detection as it may effectively allow 15.5X (7.8X+7.7X) ability to detect malignancy-associated ctDNA. Table 1. Quantification of usctDNA and mnctDNA in Plasma and Saliva Mut usctDNA (L858R) WT cfDNA usctDNA mnctDNA U/M uscfDNA mncfDNA U/M Plasma 1555 ± 326 964 ± 209 1.6X 15201 ± 1509 13261 ± 1424 Saliva 12158 ± 1731 7442 ± 1020 1.6X 340896 ± 27170 601733 ± 71573 1.1X Saliva/Plasma (fold X) 7.8 7.7 1.6X 22.4 45.3 0.6X Citation Format: Feng Li, Fang Wei, Hua-Jun He, Chien-Chung Lin, Szu-Chun Yang, Allen Wei-Lun Huang, Cheuk Ying Tang, Yong Kim, David T. Wong. Ultra-short ctDNA: A new paradigm for ctDNA liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB106.

Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2 V617F Mutation.

Precise quantification of the JAK2 V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2 V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2 V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations.

Genotyping USA laboratory-maintained isolates and European clinical isolates of Dirofilaria immitis to assess macrocyclic lactone susceptibility or resistance at predictive SNP sites using droplet digital PCR

Dirofilaria immitis is a parasitic nematode that causes cardiovascular dirofilariosis ("heartworm disease”) primarily in canids. The principal approach for mitigating heartworm infection involves the use of macrocyclic lactone (ML) for prophylaxis. Recent research has substantiated the emergence of D. immitis displaying resistance to MLs in the USA. Numerous factors, such as the mobility of companion animals and competent vectors could impact the spread of drug resistance. Genomic analysis has unveiled that isolates resistant to ML exhibit unique genetic profiles when compared to their wild-type (susceptible) counterparts. Out of the ten single nucleotide polymorphism (SNP) markers validated in clinical samples of D. immitis from the USA, four have demonstrated their effectiveness in distinguishing between isolates with varying ML efficacy phenotypes. This study explores the potential of these confirmed SNPs for conducting surveillance studies. Genotypic analysis using SNP markers emerges as a valuable tool for carrying out surveys and evaluating individual clinical isolates. Two USA laboratory-maintained isolates (Berkeley, WildCat) and twenty-five random European clinical samples of either adult worms or microfilariae (mf) pools isolated from domestic dogs, were tested by droplet digital PCR (ddPCR)-based duplex assay. This approach elucidates genetic evidence pertaining to the development of drug resistance and provides baseline data on resistance related genotypes in Europe. The data on these clinical samples suggests genotypes consistent with the continued efficacy of ML treatment regimens in Europe. In addition, this assay can be significant in discriminating cases of drug-resistance from those possibly due to non-compliance to the recommended preventive protocols.