Specific ctDNA Research Articles

LBA55 An ultra-sensitive and specific ctDNA assay provides novel pre-operative disease stratification in early stage lung cancer

LBA55 An ultra-sensitive and specific ctDNA assay provides novel pre-operative disease stratification in early stage lung cancer

High-throughput extraction on a dynamic solid phase for low-abundance biomarker isolation from biological samples

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum.

Endovascular image-guided sampling of tumor-draining veins provides an enriched source of oncological biomarkers.

Circulating tumor-derived biomarkers can potentially impact cancer management throughout the continuum of care. This small exploratory study aimed to assess the relative levels of such biomarkers in the tumor-draining vascular beds in patients with solid tumors compared to levels in their peripheral veins. Using an endovascular image-guided approach, we obtained blood samples from peripheral veins and other vascular compartments-including the most proximal venous drainage from solid tumors-from a set of nine oncology patients with various primary and metastatic malignancies. We then interrogated these samples for a panel of oncological biomarkers, including circulating tumor cells (CTCs), exosome-derived microRNAs (miRNAs), circulating tumor DNA (ctDNA) mutations, and certain cancer-related proteins/biochemical markers. We found substantially higher levels of CTCs, certain miRNAs, and specific ctDNA mutations in samples from vascular beds closer to the tumor compared with those from peripheral veins and also noted that some of these signals were altered by treatment procedures. Our results indicate that tumor-proximal venous samples are highly enriched for some oncological biomarkers and may allow for more robust molecular analysis than peripheral vein samples.

Abstract P2-08-15: Clinical, pathologic, and molecular associations of tumor mutational burden in metastatic breast cancer

Abstract Background: Tumor mutational burden (TMB) is a biomarker approved to predict response to immune checkpoint blockade (ICB) in solid tumors irrespective of their tissue of origin. However, there are limited data in patients with breast cancer and high TMB to support the use of ICB. The goal of this analysis is to describe clinical, pathological and molecular associations with TMB within a cohort of patients with metastatic breast cancer. Methods: We included patients enrolled onto an ongoing prospective study titled Individualized Molecular Analyses Guide Efforts (IMAGE)-II. Patients eligible for IMAGE-II have metastatic breast cancer of any subtype that had progressed on at least one standard-of-care therapy. Genetic profiling of tumor tissue was performed at the discretion of the treating team using one of several commercially available next generation sequencing platforms. For purposes of this analysis, only patients who underwent tissue-based Foundation Medicine analysis are included as TMB assessments are different across different platforms. Data are summarized by descriptive statistics. Linear and logistic regression analyses are conducted to evaluate the association between TMB and other clinical, pathological and molecular factors. We will present data on associations with specific mutations (i.e. ESR1, ERBB2, DNA repair, Pi3K signaling, TP53) and ctDNA TMB at a later time. Results:Of 117 patients in the IMAGE-II database, median age was 57 (range 23-86), 65% were White, 29% Black, and 6% Other. TMB data were available on 62 patients. Of those with both TMB and subtype information, 35 (70%) had ER+HER2- tumors, and 15 (30%) had ER-HER2- tumors. Median TMB was 4 mutations/megabase and ranged from 0 to 27. We did not observe significant differences in TMB in patients with ER+HER2- and those with ER-HER2- tumors (median TMB of 4 [0-27] and 5 [1-25], respectively), nor between White versus Black patients (median TMB of 4 [0-27] and 5 [0-12], respectively). However, we did observe that age was positively associated with higher TMB (p-value = 0.02). Additionally, we observed that the time between metastatic diagnosis and TMB measurement was positively associated with TMB (p-value < 0.01); this significant association was also observed in ER+HER2- patients (p-value < 0.01) but not in ER-HER2- patients. Median time to obtaining TMB since metastatic diagnosis was 1.1 (range -0.8 - 12.8) years. More lines of chemotherapy prior to TMB assessment was not observed to be associated with higher TMB. Conclusions: We observed that TMB was higher in patients who have had a longer disease course. Further research is required to understand changes in TMB over time, and how TMB is correlated with. other genomic and tumor microenvironment characteristics. A deeper understanding of TMB may help refine it as a predictive biomarker for ICB. Citation Format: Mohamed A Mohamed, Chenghuang Wang, Morgan Buckley, Jennifer Lehman, Jenna Canzoniero, Christopher D Gocke, Raquel Nunes, Ben Ho Park, Karen L Smith, Jessica Tao, Hanna Tukachinsky, Mary Wilkinson, Antonio C Wolff, Vered Stearns, Cesar A Santa-Maria. Clinical, pathologic, and molecular associations of tumor mutational burden in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-15.

Error Characterization and Statistical Modeling Improves Circulating Tumor DNA Detection by Droplet Digital PCR

Droplet digital PCR (ddPCR) is a widely used and sensitive application for circulating tumor DNA (ctDNA) detection. As ctDNA is often found in low abundance, methods to separate low-signal readouts from noise are necessary. We aimed to characterize the ddPCR-generated noise and, informed by this, create a sensitive and specific ctDNA caller. We built 2 novel complimentary ctDNA calling methods: dynamic limit of blank and concentration and assay-specific tumor load estimator (CASTLE). Both methods are informed by empirically established assay-specific noise profiles. Here, we characterized noise for 70 mutation-detecting ddPCR assays by applying each assay to 95 nonmutated samples. Using these profiles, the performance of the 2 new methods was assessed in a total of 9447 negative/positive reference samples and in 1311 real-life plasma samples from colorectal cancer patients. Lastly, performances were compared to 7 literature-established calling methods. For many assays, noise increased proportionally with the DNA input amount. Assays targeting transition base changes were more error-prone than transversion-targeting assays. Both our calling methods successfully accounted for the additional noise in transition assays and showed consistently high performance regardless of DNA input amount. Calling methods that were not noise-informed performed less well than noise-informed methods. CASTLE was the only calling method providing a statistical estimate of the noise-corrected mutation level and call certainty. Accurate error modeling is necessary for sensitive and specific ctDNA detection by ddPCR. Accounting for DNA input amounts ensures specific detection regardless of the sample-specific DNA concentration. Our results demonstrate CASTLE as a powerful tool for ctDNA calling using ddPCR.

The Prognostic and Therapeutic Value of the Mutational Profile of Blood and Tumor Tissue in Head and Neck Squamous Cell Carcinoma.

BackgroundThe purpose of this study was to explore the genomic landscape of head and neck squamous cell carcinoma (HNSCC) in circulation (circulating tumor DNA [ctDNA]) and tumor (tumor tissue DNA [tDNA]) and understand the implications of ctDNA sequencing for prognosis and precision oncology treatments.Materials and MethodsThis is a retrospective review of 75 patients with HNSCC for both tDNA and ctDNA. Results were analyzed for concordance between tDNA and ctDNA and for their individual and combined association with demographics, survival, and presence and extent of disease at last visit (DLV).ResultsThe five most frequently altered genes were TP53, CDKN2A, TERT, BRCA2, and NOTCH1. Twenty percent of patients had NOTCH1 alterations in tDNA, with none found in ctDNA. Concordance among altered genes was 13.0%, and 65.3% of patients had actionable ctDNA alterations. ctDNA alterations were significantly associated with decreased overall survival (OS) and presence and extent of DLV. In DNA repair genes, alterations in ctDNA alone and combined with tDNA were significantly associated with decreased OS and presence of DLV. Similar significant associations were found in TP53 for ctDNA alone and combined with tDNA. DNA repair gene alterations in ctDNA and unique ctDNA alterations within partially concordant genes were significantly associated with decreased OS in multivariate analysis.ConclusionThis study illustrates the circulating and tumor genomic profile in the largest HNSCC cohort to date, underscoring the potential utility of ctDNA in prognostication and precision oncology treatment. For the first time, the presence of ctDNA alterations and specific ctDNA sequencing results were shown to be significantly associated with poor prognosis in HNSCC.Implications for PracticeThe use of precision genomic targeted therapies in head and neck squamous cell carcinoma (HNSCC) lags behind many other cancers, and poor survival in advanced stages indicates the urgent need for improved treatment options. This exploratory analysis of circulating tumor DNA (ctDNA) and tumor tissue DNA (tDNA) sequencing in the largest cohort to date of patients with HNSCC provides a novel depiction of the ctDNA genome, with two thirds of patients having actionable ctDNA alterations. This study reports for the first time the prognostic value of ctDNA sequencing, with the presence of ctDNA alterations, specific ctDNA alterations in DNA repair genes and TP53, and unique ctDNA alterations within partially concordant genes predicting poor survival.

Private Payer and Medicare Coverage for Circulating Tumor DNA Testing: A Historical Analysis of Coverage Policies From 2015 to 2019.

Clinical adoption of the sequencing of circulating tumor DNA (ctDNA) for cancer has rapidly increased in recent years. This sequencing is used to select targeted therapy and monitor nonresponding or progressive tumors to identify mechanisms of therapeutic resistance. Our study objective was to review available coverage policies for cancer ctDNA-based testing panels to examine trends from 2015 to 2019. We analyzed publicly available private payer policies and Medicare national coverage determinations and local coverage determinations (LCDs) for ctDNA-based panel tests for cancer. We coded variables for each year representing policy existence, covered clinical scenario, and specific ctDNA test covered. Descriptive analyses were performed. We found that 38% of private payer coverage policies provided coverage of ctDNA-based panel testing as of July 2019. Most private payer policy coverage was highly specific: 87% for non-small cell lung cancer, 47% for EGFR gene testing, and 79% for specific brand-name tests. There were 8 final, 2 draft, and 2 future effective final LCDs (February 3 and March 15, 2020) that covered non-FDA-approved ctDNA-based tests. The draft and future effective LCDs were the first policies to cover pan-cancer use. Coverage of ctDNA-based panel testing for cancer indications increased from 2015 to 2019. The trend in private payer and Medicare coverage is an increasing number of coverage policies, number of positive policies, and scope of coverage. We found that Medicare coverage policies are evolving to pan-cancer uses, signifying a significant shift in coverage frameworks. Given that genomic medicine is rapidly changing, payers and policymakers (eg, guideline developers) will need to continue to evolve policies to keep pace with emerging science and standards in clinical care.

Sensitive and selective ctDNA detection based on functionalized black phosphorus nanosheets

Circulating tumor DNA (ctDNA) identification is one of the most meaningful approaches towards early cancer diagnosis. However, effective and practical methods for analyzing this emerging class of biomarkers are still lacking. In this work, a biosensor based on nitrophenyl functionalized black phosphorus nanosheets (NP-BPs) is fabricated for sensitive and selective detection of ctDNA. In this work, a nitrophenyl functionalized black phosphorus nanosheets (NP-BPs) biosensor is fabricated for sensitive and selective detection of ctDNA. Due to the successful nitrophenyl functionalization, the NP-BPs biosensor shows higher quenching efficiency and stronger affinity towards single-stranded DNA (ssDNA), as compared with double-stranded DNA (dsDNA). Therefore, the NP-BPs biosensor exhibits 5.4-fold fluorescence enhancement when dye-labelled ssDNA probe forms dsDNA in the presence of its specific ctDNA target. This biosensor exhibits a detection limit of 50 fM and a wide linear detection range of 50 fM-80 pM, provides reliable readout in a short time (15 min). Moreover, the NP-BPs-based biosensor could be applied to discriminate single nucleotide polymorphisms in clinical serum samples. It is envisioned that the NP-BPs-based sensing platform has great potentials in early cancer diagnosis and monitoring cancer progression.

Applications of cell-free circulating tumor DNA detection in EGFR mutant lung cancer.

Analyses of cell-free tumor DNA (ctDNA) have provided a non-invasive strategy for cancer diagnosis, the identification of molecular aberrations for treatment identification, and evaluation of tumor response. Sensitive and specific ctDNA sequencing strategies have allowed for implementation into clinical practice for the initial genotyping of patients and resistance monitoring. The specific need for EGFR mutation detection for the management of lung cancer patients has been an early imperative and has set the stage for non-invasive molecular profiling across other oncogenic drivers. Ongoing efforts are demonstrating the utility of ctDNA analyses in the initial genotyping of patients, the monitoring resistance clones, and the initial evaluation of response.

Abstract P2-03-05: TP53 mutations in circulating tumor DNA associate with klebsiella pneumoniae inbreast cancer

Abstract Background Breast cancer is a heterogeneous disease with different molecular pathological types and different clinical manifestations. Liquid-based analysis of tumor heterogeneity in circulating tumor DNA (ctDNA) can be used for cancer detection, monitoring disease progression, etc. The gut microbiome has emerged as an important mediating factor of breast cancer. Dysbiosis may contribute to the development of breast cancer. In this study, we aimed to identify risk-associated microbiome and whether some of specific microbiomes correlate with tumor-specific mutation burdens and disease progression in Taiwan breast cancer patients. Methods A total of 141 normal female subjects and 52 breast cancer patients were enrolled randomly. The study was approved from the Internal Review Board of Kaohsiung Medical University Hospital and informed consents were obtained from each subject. The Oncomine™ breast cfDNA assays with 10-gene panel of 152-associated mutational hotspot loci was performed to detect circulating breast tumor-derived DNA (ctDNA). The microbiota composition in fecal samples was analyzed using 16S ribosome RNA gene (V3-V4 region) amplicon sequencing on Illumina Miseq platform. 16S rRNA sequencing data were analyzed using CLC genomics workbench (Qiagen, Germany) and MeTaxon software (DNArails Co., Ltd, Taipei, Taiwan, ROC). Results Multiple logistic regression was used to compare the differences of druggable gene mutations and 59.2% of patients harbored at least one somatic mutation. Compared to TP53, the odds of mutations of PIK3CA, ERBB2, AKT, KRAS, and SF3B1 were 94.4%, 98.5%, 99.3%, 99.3%, and 99.7% were significantly lower than those of TP53 (all p<0.001), after adjusting for tumor staging. As compared with stage I&II, the risk of harboring gene mutations was significantly higher in stage III (OR=9.519; 95% CI: 2.862-31.661), after adjusting for the effect of mutation type. Further investigation of the gut microbiota in breast cancer patients and normal controls showed that patients had an increased fecal microbiota composition (β-diversity) (ANOSIM, R2=0.0799, p=0.01 for Bray Curtis distances) but a lower α-diversity (pChao1=0.02) when compared with control women. Patients in stage I&II group had higher relative abundance of Streptococcus, Pasteurellaceae, Klebsiella pneumoniae, Blautia and Lachnospiraceae, as compared with control (p=0.003, <0.001, 0.001, <0.001 and 0.01).The ratio of increased microbial group (Streptococcus, Pasteurellaceae, Klebsiella pneumoniae, and Blautia) to decreased one (Sphingobacteriaceae and Akkermansia muciniphila) could obtain a score to discriminate stage I&II from normal control (AUC=0.783, p<0.001). Intriguingly, Lachnospiraceae wasincreased along with stage progression. In addition, the patients with TP53 mutations had higher levels of Klebsiella and those ones with PIK3CA mutations had higher levels of Lachnospiraceae and Sphingobacteriaceae. Blautia was increased when patients presented with both TP53 and PIK3CA mutations. More importantly, the proportion of TP53 mutations was higher and specially enriched with Klebsiella pneumoniae. Conclusion Our results showed that several microbiomes were significantly changed in breast cancer patients and the ratio of increased microbial group to decreased one could discriminate stage I&II from normal control. In addition, these microbiomes were correlated to specific ctDNA mutation patterns. Mutations of TP53 was associated with the enrichment of Klebsiella pneumoniae which was highly represented in breast cancer. Dysbiosis of specific gut microbiome may serve as novel biomarkers for early detection and prevention of breast cancer. The mechanisms underlying specific association between gut microbiome and gene mutations require further investigations. Citation Format: Ming-Feng Hou, Chih-Po Chiang, Chuan-Hsin Chang, Wei-Ting Lien, Ming-Je Yang, Yao-Tsung Yeh. TP53 mutations in circulating tumor DNA associate with klebsiella pneumoniae inbreast cancer [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 P2-03-05.

Sequenced circulating tumor (ct) DNA to detect the molecular landscape in advanced GIST.

11036 Background: Molecular (mol) characteristics-guided precision therapy has well-established utility in GIST management. The GIST mol landscape is poorly represented by selective tumor biopsy (bx) material, especially in metastatic and treatment-refractory GIST. ctDNA can overcome some of the limitations of selective tissue bx specimens and provide comparable or greater mol information. ctDNA represents an emerging mol biomarker in advanced (adv) GIST. Methods: Next generation sequencing analyses (MSK-IMPACT or custom-capture GIST specific panel) were performed on ctDNA prospectively collected from patients (pts) with adv GIST. MSK-IMPACT was performed on tumor material (archival tissue or paired tissue bx specimens taken in parallel with ctDNA collection from clinical trial participants). The primary objective was to determine the concordance between the mol landscape of GIST identified by sequenced tumor tissue versus ctDNA. Secondary objective was to correlate characteristics of sequenced ctDNA with standard measurements of response assessment. Results: 47 ctDNA samples collected from 25 pts with adv GIST were sequenced using MSK-IMPACT ( > 400 genes). MSK-IMPACT was performed on archival tumor material. Sequenced ctDNA detected the mutational spectrum of tumor tissue in 48% of cases. In 50% of these cases, ctDNA identified novel treatment-resistance mutations not previously identified in the archival tissues. To optimize sensitivity of detection, a custom-designed GIST specific ctDNA sequencing panel was developed incorporating 19 of the most commonly altered genes observed from MSK-IMPACT analysis performed on > 75 imatinib-refractory adv GIST tumor samples. 52 ctDNA samples collected from 30 pts with paired tumor bx samples have been sequenced using the custom-capture GIST panel. The tumor bx samples have been sequenced using MSK-IMPACT. Results are pending and will be presented. A separate cohort of ctDNA samples serially collected in the setting of a prospective clinical trial are being sequenced. The mol results with correlated treatment response by RECIST will also be presented. Conclusions: ctDNA could detect a broad spectrum of mol heterogeneity and potentially be used as a biomarker to guide precision therapy in adv GIST. Further research is necessary to determine the optimal ctDNA sequencing assay and the appropriate clinical setting to utilize in GIST.

Analytical validation of a tumor-agnostic integrated multianalyte circulating tumor DNA (ctDNA) assay in early-stage cancer.

3057 Background: ctDNA sequencing has been rapidly adopted for the identification of targetable somatic alterations (alts) in patients with advanced cancers. However, early stage disease detection has been hindered by low levels of ctDNA in circulation and the presence of confounding non-tumor-related somatic alts. We developed and validated a ctDNA assay that combines somatic and epigenomic signals to detect early stage tumors without tumor tissue or white blood cells (WBC). Methods: Using a single input sample, our assay integrates the sensitive detection of genomic alts with quantification of epigenomic signals associated with cancer. Non-tumor alts (e.g., clonal hematopoiesis of indeterminate potential; CHIP) are excluded using a newly developed bioinformatic classifier. To assess analytical sensitivity, specificity, and positive and negative reproducibility, we tested 337 clinical and contrived samples. Results: Clinical specificity was determined using 80 plasma samples from 50-75 year old presumptive cancer-free donors, and resulted in a single false positive (99% specificity). Analytical sensitivity (limit of detection) was established using a dilution series of 4 different late stage CRC pts tested in triplicate at a clinically relevant DNA input (30 ng) across multiple batches. 100% sensitivity was maintained even at the lowest tested level (estimated 0.1% tumor level). Positive/negative reproducibility was assessed by testing triplicates of diluted late stage samples and age-matched healthy donors, respectively, across different cfDNA inputs, and multiple reagent lots. Both positive and negative calls were 100% concordant across all replicates. Independent estimation of tumor levels from epigenomic or genomic signals produced highly concordant results (correlation r-value: 0.82, p-value: 3e-16). Conclusions: We designed and validated a highly specific ctDNA assay that integrates both genomic and epigenomic signals to allow for accurate and quantitative tumor level detection in early stages of the disease without requiring tumor tissue or WBC.

Molecular profiling by circulating tumor DNA (ctDNA) and benefit from anti-PD-1 in HCC.

e15679 Background: Molecular profiling has defined actionable mutations in HCC, and has the potential to be used for selection of targeted therapies, as well as for the characterization of predictive biomarkers from approved treatments. Noninvasive strategies are critical to HCC given the challenge of obtaining liver biopsies. We investigated whether profiling by ctDNA could provide predictive and/or prognostic information for HCC patients (pt) treated with immune checkpoint inhibitors. Methods: We analyzed blood samples from 22 HCC pt who underwent treatment with anti-PD-1 using comprehensive genomic testing of ctDNA with a commercially-available platform (Guardant Health, CA). Demographic and treatment data were retrospectively collected with the goal of correlating treatment outcomes and drug response (by imaging and/or AFP) with molecular abnormalities. Results: 17/22 (77.3%) were men; median age was 66 years. 21 patients received nivolumab and 1 received pembrolizumab. 9 were HCV positive and 5 were HBV positive. 15/22 patients had > 1 alteration identified. The median number of alterations/pt was 3 (range, 1-8). TP53 was the common altered gene (n = 11) followed by CTNBB1 (n = 8) , TERT (n = 5) KRAS (n = 3) , GNAS (n = 2). Mutations were also seen (n = 1) in KIT, PIK3CA, PTEN, EGFR, NTRK, FGFR2 among others. 6 pt (27.3%) had AFP response and 8 (36.4%) achieved disease control > 12 weeks. Mutations involving KIT, PIK3CA and PTEN were associated with shorter progression-free (PFS) (p < .001 for all) and overall survival (OS) (p = .028 for all), whereas GNAS mutation was associated with shorter PFS (p = 0.019) but not OS. No differences in OS or PFS was observed for other alterations, including the presence of CTNNB1 mutation. There were no correlations between specific alterations and objective tumor response (either by imaging or AFP). 32% of pt were progression-free at 6 months. Median OS was not reached, and 62% were alive after 1 year. Conclusions: Identifying non-invasive predictive biomarkers of benefit to immunotherapy is a priority in HCC. Our data suggest that specific ctDNA alterations can provide predictive information for survival (OS and PFS) on immune checkpoint inhibitors. Further larger studies are warranted for confirmation.

Circulating tumor DNA (ctDNA) during and after neoadjuvant chemotherapy and prior to surgery is a powerful prognostic factor in triple-negative breast cancer (TNBC).

594 Background: TNBC, the most aggressive form of breast cancer, is treated primarily with chemotherapy, even before surgery (neoadjuvant chemotherapy or NAC). The prognosis and need for adjuvant therapy depends greatly on the tumor response assessed by pathology (pCR). Highly sensitive and specific ctDNA assays have been shown to be of prognostic value in the metastatic settingbut not yet in earlier settings. Methods: Tissue was collected from 26 Q-CROC-03 clinical trial TNBC patients before, during and after NAC, prior to surgery. Whole exome sequencing on tumor tissues was used to select single nucleotide variants with high allele frequency (VAF), prioritizing TP53, to generateindividual digital droplet PCR (ddPCR) assays. An average of 5 variants (range 1-12) per patient were tested, for a total of 121 variants. A detection threshold was defined for each variant from a pool of normal controls. Median follow-up was 55 months. Results: ctDNA was detectable in 96% of patients at baseline, but 20% of the 121 variants were not detectable at any time point. At baseline, the mean VAF of all analyzed variants, but not of TP53 variants alone, was significantly correlated (p < 0.05) with tumor factors (tumor size, stage, grade, nodal status before and at surgery, RCB score) but not with patient age or BRCA1/2 mutation status. 87 variants (74%) were detected at baseline and their VAF fell by 86% after 1 cycle of chemotherapy (T1). The detection of ctDNA at T1 was associated with DFS (p = 0.027) while the detection of ctDNA at the last post-chemotherapy pre-surgery time point (T4) was strongly associated with pathological complete response (pCR) and both DFS (p = 0.013) and OS(p = 0.006). At this time point, 5 of 41 variants (12%) were detected in pCR patients vs 42 of 80 (53%) in non-pCR, while only 6 of the 15 (40%) non-pCR patients had detectable TP53 variants. Interestingly, for variants detected at baseline, the positive predictive value of T4 ctDNA for disease recurrence was 69%, similar to that of non-pCR, while the negative predictive value of no ctDNA at T4 was 89% for disease recurrence vs 80% for pCR. Conclusions: ctDNA detection after NAC prior to surgery is strongly predictive of disease-free survival and overall survival and is comparable to pCR as a prognostic factor in our cohort (NCT01276899).

Molecular characterization and monitoring of patient ctDNA in phase I study of H3B-6545 in ER+ MBC.

1052 Background: Because of lack of effective treatment in endocrine resistant metastatic breast cancer (MBC), we developed H3B-6545, a novel selective ERa covalent antagonist, capable of irreversibly inactivating both wild-type and mutant ERa. The aims of this study are to 1) characterize hotspot mutation profiles in heavily pretreated MBC and correlate ESR1, PIK3CA and AKT1 mutations in plasma vs tumor tissue 2) determine if mutations in ESR1 or PIK3CA predict response to H3B-6545 and 3) evaluate if longitudinal tracking of ctDNA correlates with response to H3B-6545. Methods: Fresh plasma samples were collected at baseline (predose), cycle 1 day 15 (C1D15), C2D1, C3D1 and every 8 weeks thereafter with a final sample collection at disease progression. At baseline, BEAMing digital PCR was used to evaluate hotspot mutations in ESR1, PIK3CA and AKT1. Patient specific ctDNA mutations were subsequently monitored by ddPCR. Baseline tumor biopsies were subjected to a targeted Next Generation Sequencing (NGS) panel to identify hotspot mutations. Results: In 77% of patients (30/39), mutations were detected at baseline by the BEAMing assay and of those, 21/39, 16/39 and 3/39 had mutations in ESR1, PIK3CA and AKT1, respectively. 20% (9/39) of patients exhibited co-mutations in ESR1 and PIK3CA. In 60% (9/15) of patients, DNA mutations identified by the plasma BEAMing assay were also detected in the tumor biopsy whereas; DNA mutations found in tissue were also detected in plasma in 86% (12/14) of cases. Serial ctDNA monitoring revealed that in patients with confirmed partial responses (3/3), ctDNA levels were undetectable by C2D1. In contrast, ctDNA levels increased from baseline in 3/4 patients with progressive disease. Exploration of ctDNA ratios (day 15/baseline and day 30/baseline) and correlations of PIK3CA and ESR1 mutations with response to H3B-6545 will be presented. Conclusions: ctDNA is a reliable sample type for assessing ESR1, PIK3CA, and AKT1 mutations in MBC, overcoming the challenges of obtaining biopsies in the metastatic setting. In addition, ctDNA dynamics may be a useful tool to monitor the efficacy of H3B-6545. Clinical trial information: NCT03250676.

Ultrasensitive Detection of Circulating Tumor DNA of Lung Cancer via an Enzymatically Amplified SERS-Based Frequency Shift Assay.

Circulating tumor DNA (ctDNA) is a promising noninvasive biomarker for the early diagnosis of cancers. However, it is challenging for accurate and sensitive detection of pico-to-femtomolar serum concentration of ctDNA, especially in the presence of its analogues that produce strong background noise. Herein, a DNA-rN1-DNA-mediated surface-enhanced Raman scattering frequency shift assay is developed, which enables sensitive detection of ctDNA with one single base pair mutation (KARS G12D mutation) from the normal ones (KARS G12D normal) of lung cancer. This sensing platform features in both the designed hairpin DNA-rN1-DNA probe for specific ctDNA recognition and the employed RNase HII enzyme that specifically hydrolyzes the DNA-rN1-DNA/ctDNA hybrid and thus allows ctDNA recycling in the system to realize signal amplification. The detection system shows sub-femtomolar-level sensitivity in the phosphate-buffered saline solution and is demonstrated to function well in both fetal bovine serum and human physiological media. In particular, the sensitive assay of ctDNA in serum samples from lung cancer patients is achieved, suggesting its high potential applications in clinical settings for early diagnosis and prognosis of lung cancer.

Abstract P4-01-18: Correlation between circulating tumor DNA (ctDNA) alterations and circulating tumor cells (CTC) uncovers new mechanisms of metastasis for patients with metastatic breast carcinoma (MBC)

Abstract Introduction:Novel molecular diagnostics including CTCs and ctDNA have been proved to predict disease metastasis and survival. However, the frequency of detection of actionable mutations using CTCs and ctDNA is variable based upon tumor related factors and diagnostic platform sensitivity. Herein, we evaluated a novel NGS technology in the ability of detecting driver and clonal genomic abnormalities in samples from MBC patients, and compared ctDNA alterations with CTCs and CTC-cluster. This study demonstrated several novel correlation between some specific ctDNA alterations and CTCs or CTCs related biomarkers, which opened new insight on mechanisms of metastasis for MBC. Methods: This study included 52 samples from 26 patients with stage III/IV BCa treated at NMH (2016-2017) and who received standard systemic treatments based on disease subtypes. Whole blood samples (7.5ml/each) were used for CTC enrichment and enumeration in FDA approved CELLTRACKS ANALYZERII® System (Menarini). ctDNA from clinical plasma samples was analyzed by using PredicinePLUS, a NGS-based assay (Predicine Inc) with a 180-gene panel for genomic alterations mutations. Results of CTCs and ctDNA alterations were linked to clinical database. Matched pairs variations between CTCs and ctDNA alterations was compared by Wilcoxon signed-ranks test and Kruskal-Wallis test. Results: Genomic Alterations (SNVs, Indels and copy number variations) were detected on 52 genes by PredicinePLUS assay. All samples (100%) demonstrated at least 1 somatic alterations. There were 75 mutations detected within 29 genes, and the variant frequency of mutated genes ranges from 0.11% to 68.56%. Increased CTCs were highly significantly correlated with genomic alterations in the genes (wild type vs alterations) including GATA3 (8vs 37), ESR1 ( 2.5 vs 41.3), CDH1 (3.5 vs 50.5) and CCND1 (4 vs 120) (P<0.01). Decreased CTCs were correlated with alterations of CDKN2A (20.5 vs 0) (P=0.025). CTC-cluster appear associated predominantly with alterations of CDH1 (P=0.0018), CCND1 (P=0.008) and BRCA1 (P=0.04). Furthermore, in HER positive CTCs group, ERBB2 mutations caused increased CTCs in compared with ERBB2 wild type (0 vs 5), when CCND1, CDKN2A, GATA3 and TP53 alterations were associated with increase of HER2 negative CTCs. Conclusions: By using the novel diagnostic platform with the ability to identify ctDNA mutation and copy number variation, this study demonstrated several novel genes alterations which were highly correlated with CTCs, CTC-cluster and HER2. Some genes (CCND1 and CDH1) got involved into the changes on both CTCs and CTC-cluster, when some genes (CCND1, CDKN2A, ESR1 and GATA3) were related with change of CTCs and HER2 expression. Correlation of CTCs and ctDNA can be reliably and routinely used as non-invasive method for monitoring disease metastasis and predict the prognosis in MBC in clinic. Citation Format: Davis A, Zhang Q, Gerratana L, Zhang Y, Flaum L, Shad A, Behdad A, Gradishar W, Platanias L, Cristofanilli M. Correlation between circulating tumor DNA (ctDNA) alterations and circulating tumor cells (CTC) uncovers new mechanisms of metastasis for patients with metastatic breast carcinoma (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-18.

Monitoring melanoma recurrence with circulating tumor DNA: a proof of concept from three case studies.

BackgroundA significant number of melanoma patients experience recurrence to distant sites, despite having had surgical treatment of the primary lesion, with curative intent. Monitoring of patients for early evidence of disease recurrence would significantly improve management of the disease, allowing timely therapeutic intervention. Circulating tumor DNA (ctDNA) is becoming a well-recognized biomarker for monitoring malignancies and has, in a few studies, been shown to signify disease recurrence earlier than conventional methods.MethodsWe performed a retrospective analysis of plasma ctDNA using droplet digital PCR (ddPCR) in 30 primary melanoma patients with tumors harboring BRAF, NRAS or TERT promoter mutations. Mutant specific ctDNA, measured during clinical disease course, was compared with disease status in patients with confirmed disease recurrence (n = 3) and in those with no evidence of disease recurrence (n = 27).ResultsMutant specific ctDNA was detected in all three patients with disease recurrence at the time of clinically confirmed progression. In one case, plasma ctDNA detection preceded clinical identification of recurrence by an interval of 4 months. CtDNA was not detected in patients who were asymptomatic and had no radiological evidence of recurrence.ConclusionsThis study demonstrates promising results for the use of ctDNA as an informative monitoring tool for melanoma patients having undergone tumor resection of an early stage primary tumor. The clinical utility of ctDNA for monitoring disease recurrence warrants investigation in prospective studies as it may improve patient outcome.

Methylated circulating tumor DNA as a potential marker of PARP inhibitor efficiency in BRCA mutated ovarian cancer patients.

5540Background: The liquid biopsy has proven to be an excellent material for analysis of different circulating tumor markers, among which mutated tumor DNA (ctDNA) ranks high. Cancer specific ctDNA...

Very early molecular marker of tumor response to PD-1 inhibition in plasma of patients with advanced non-small cell lung cancer (NSCLC).

3030 Background: Nivolumab shows a durable tumor response in 20 % of advanced NSCLC patients. While a PD-L1 immunohistochemistry complementary diagnostic has been approved, there is a need to identify a predictive biomarker with greater diagnostic accuracy. Methods: In an ongoing study of patients, treated with nivolumab every 2 weeks for advanced recurrent NSCLC and a known KRAS mutation in the primary tumor, liquid biopsies were taken by a simple blood draw at baseline (before treatment), 1, 2, 4, 6 and thereafter every 12 weeks until disease progression. Circulating tumor DNA (ctDNA) was extracted from cell free plasma and tested with a specific digital droplet PCR (ddPCR)-KRAS G12/G13 screenings assay (BIORAD). Mutation levels in plasma were compared with tumor response on CT scans by RECIST v.1.1. CT scans were performed at baseline and thereafter every 6 weeks until disease progression. Results: Patient characteristics are shown in table 1. In this prospective analysis specific patterns could be demonstrated in plasma ctDNA testing of KRAS mutation levels. All 4 responders showed an increase of mutant copies/ml plasma in the first week followed by a clear drop to non-detectable KRAS mutations at all CT evaluated tumor responses. The 2 non-responders however, did not show the increase in mutant copies/ml at one week and showed a gradual increase in mutant copies/ml in the following weeks. Conclusions: Specific plasma ctDNA testing seems a very promising early biomarker (1 week) for tumor response to PD-1 therapy. [Table: see text]