ctDNA Levels Research Articles

MATRIX plasma-in-plasma contrived samples assessment and detection of MRD using the Personalis NeXT Personal assay.

e15056 Background: Detection of circulating tumor DNA (ctDNA) has been shown to correlate with clinical outcome of oncology patients. Molecular residual disease (MRD) profiling by ctDNA is being rapidly deployed. Current tumor-informed MRD tests assess a relatively small number of personalised variants, placing limits on their detection sensitivity and ultimately leading to false-negative results due to insufficient assay limit of detection (LOD). Here we report a performance evaluation of the Personalis NeXT Personal tumor-informed MRD assay, using a novel design for contrived samples that are highly commutable to clinical samples, enabling robust assessment of these emerging MRD assays. Methods: Our novel samples were built from commercially acquired matched tumor and plasma samples, across different indications. The cancer plasma was diluted into a background of healthy donor plasma to create the 72 plasma-in-plasma samples in each study (total of 144). Personalis performed WGS on the matched solid tumor and normal samples from each patient. ~1800 somatic variants were selected for each patient. A personalized panel was designed and used to enrich for the selected targets in the individual plasma samples. An aggregated positive variant score was evaluated to determine the presence of ctDNA in each plasma sample. Results: MATRIX 1 panels averaged 1863 variants (range: 1818 to 1888) selected for MRD tracking. With 152 clinically relevant variants and additional assay components, an average of 2206 variants were assessed per patient. MATRIX 2 panels averaged 1836 variants (range: 1819 to 1867) selected for MRD tracking. With 2130 clinically relevant variants they averaged 4094 variants assessed per patient. In the MATRIX 1 study, tumor signal was detected in all analyzed samples, including 9 samples at 0.002% tumor plasma dilution, with an assay sensitivity of 100%. The MATRIX 2 study showed tumor signal detected in 14 of 16 samples at 0.001% tumor plasma dilution. The 2 false negatives were reported at the 0.001% tumor plasma dilution. Thus, the assay sensitivity was 87.5% at the lowest dilution (0.001%) and 96.8% overall. Conclusions: The MATRIX plasma-in-plasma approach is a robust option for assessing the sensitivity of MRD assays to low levels of ctDNA. This approach addresses all parts of an MRD assay for both the plasma and tumor/normal tissues, and is the closest thing to real clinical samples while allowing direct interrogation of sensitivity. Additionally, it allows assessment of any genomic or epigenomic biological feature. The Personalis NeXT Personal assay shows ultra-high sensitivity with reproducible data down to the 1-3 PPM range, and expansion of the personalized panel fixed clinical content allows tracking of more driver and resistance mutations with no impact to the MRD sensitivity.

A phase 2/3 trial in progress on tebentafusp as monotherapy and in combination with pembrolizumab in HLA-A*02:01+ patients with previously treated advanced non-uveal melanoma (TEBE-AM).

TPS9594 Background: Tebentafusp is a bispecific (gp100 x CD3) ImmTAC that can redirect T cells to target gp100+ melanoma cells. In a Phase (Ph) 3 trial, tebentafusp demonstrated an overall survival (OS) benefit (HR 0.51) compared to investigator's choice (IC) in first line HLA-A*02:01+ patients with metastatic uveal melanoma (mUM). Gp100, which has limited expression in normal cells, is overexpressed in melanoma, including skin melanoma, supporting the investigation of tebentafusp in non-uveal melanoma. In a first-in-human trial, tebentafusp monotherapy demonstrated a promising 1-year OS (~74%) in anti-PD(L)1 naïve HLA-A*02:01+ patients with metastatic cutaneous melanoma (mCM). In a subsequent Ph1 trial, tebentafusp combined with anti-PDL1, with or without anti-CTLA4, demonstrated a 1-year OS of 75% in patients with mCM who progressed on prior anti-PD(L)1 therapy, which compares favorably with recent benchmarks (1-year OS 38%-57%) in similar patient populations.1-3 The promising activity of tebentafusp as monotherapy and in combination with anti-PD(L)1 agents in advanced melanoma (AM) provides the rationale to conduct a Ph2/3 trial of tebentafusp (TEBE-AM; NCT05549297) in patients who have progressed on standard of care therapies. In mUM trials, RECIST response underestimated the OS benefit of tebentafusp, whereas a strong association was shown between OS and early reduction in ctDNA levels relative to baseline.4 As an early measure of changes in tumor burden, ctDNA reduction is included as an innovative dual primary endpoint with OS for the Ph2 part of this study. Methods: TEBE-AM is a multicenter, open-label, seamless Ph2/3 study in HLA-A*02:01+ patients with non-uveal AM who have progressed on prior anti-PD(L)1, received prior ipilimumab, and a prior BRAF/MEK inhibitor regimen if actionable BRAF mutation is present. Patients are randomly assigned (1:1:1) to receive tebentafusp monotherapy, tebentafusp combined with pembrolizumab, or IC. In the IC arm, patients may enroll in clinical trials of investigational agents, receive local standard of care, or receive best supportive care, while being followed for key endpoints. Randomization into the Ph3 portion will commence immediately following completion of accrual into the Ph2 portion. Efficacy from the Ph2 portion may inform changes to the Ph3 design. Primary endpoints are ctDNA reduction relative to baseline and OS in Ph2 and OS in Ph3. Ph2 enrollment opened in Jan 2023 in the US and is expected to open in other countries mid-2023. Clinical trial registration: NCT05549297. 1. Zimmer L, et al. Eur J Cancer 75:47–55, 2017. 2. Silva IPD, et al. J Clin Oncol 38:10005–10005, 2020. 3. Arance AM, et al. J Clin Oncol 39:S9504, 2021. 4. Carvajal RD, et al. Nat Med 28:2364-2373, 2022. Clinical trial information: NCT05549297 .

Urine DNA model to predict response to neoadjuvant immune checkpoint inhibitors combined with chemotherapy in muscle-invasive bladder cancer.

e16594 Background: Neoadjuvant immune checkpoint inhibitors (ICIs) combined with chemotherapy show promise in the treatment of muscle-invasive bladder cancer (MIBC). However, not all patients benefit from the treatment. Urine can overcome shortcomings of tissue-based biomarkers to guide treatment decisions. Methods: Thirty-Three patients were recruited in this study and they received three cycles of tislelizumab 200 mg intravenously on day 1 plus nab-paclitaxel 200 mg intravenously on day 2 every three weeks, followed by surgical treatment. Acornmed 808 panel and shallow whole-genome sequencing were performed to predict the efficacy based on the urine DNA. Results: The urine ctDNA levels (highest variant allele fraction) were significantly lower in responder (CR and PR ) patients than this in non-responder (SD and PD) patients (p < 0.001). In addition, a decrease in chromosomal instability number (CIN) occurred in responding tumors (p = 0.028). Furthermore, ROC curves were used to compare the sensitivity and specificity of efficacy prediction, including the urine ctDNA level and CIN. In the ROC analysis, urine ctDNA level setting with an area under the ROC curve (AUROC) of 0.781 (95% CI 0.603 - 0.905, p = 0.005), achieving 100% sensitivity and 54.6% specificity in efficacy prediction. The ROC curve for CIN achieved a sensitivity of 68.2% and specificity of 72.7% (AUC = 0.731; 95% CI: 0.549-0.870, p = 0.013). Combination urine ctDNA level and CIN achieved a sensitivity of 100%, specificity of 63.6% (AUC = 0.806; 95% CI: 0.631-0.922, p = 0.002), positive predictive value (PPV) of 0.846, and negative predictive value (NPV) of 1.000. Conclusions: The classifier provides a framework for novel biomarker discovery and for optimizing treatment and surveillance in next-generation clinical trials.

Single-stranded linear amplification library preparation method for highly sensitive and low input amount ctDNA detection.

e15096 Background: Detection of circulating tumor DNA (ctDNA) or minimum residual disease (MRD) through somatic variant identifications in plasma samples has emerged as a promising approach for monitoring the clinical outcomes after surgical resections or other therapies. However, due to very low ctDNA levels in the early-stage cancers and tumor remissions after therapies, or low plasma volumes and DNA degradation caused low qualities in clinical practice, to sensitively identify somatic mutations with low allele frequencies in those samples remains a challenging technology task. Methods: In this study, we developed a single-stranded, linear amplification based library preparation sequencing (SLA-Seq) method. In the experimental workflow, double stranded molecular templates will be converted into single strands to construct libraries, thus minimize the loss of irregular double stranded DNA molecules. Adapter ligation was carried out in two steps. During the first step, P7 adapters with unique molecular identifiers(UMI) were ligated to the 3’ end of the molecular templates, and the DNA molecules were amplified by linear amplification to improve the DNA utilization efficiency. In the second step, P5 adapters were ligated to the linear amplified PCR products, then amplified by the pre-PCR. The pre-library was enriched by hybridization and magnetic-beads capturing and then subjected to high-throughput sequencing. 30ng of the cfDNA reference standard samples containing mutations of 0.3% and 0.1% allele frequencies were used to validate the performance. Results: In the SLA-Seq method, adapter sequences were carefully optimized to eliminate adapter dimers and thus improved the DNA ligation efficiency. Compared with the double-stranded library preparation method, SLA-seq showed overall higher effective depths after removing duplications if the same start amount of the DNA was used, which indicated that the higher DNA utilization rate was achieved. To evaluate the performance, we applied SLA-Seq on 11 reference standard samples with only 30ng start amount DNA. Among them, 6 samples were diluted to 0.3% allele frequency and 5 samples were diluted to 0.1% allele frequency. Totally they had 24 and 35 mutations, respectively. The sensitivity at the 0.3% allele frequency was 100% (24/24) and it was 94.29% (33/35) at the 0.1% mutation level, which also showed the higher sensitivity achieved by SLA-Seq than double-stranded library preparation method. Conclusions: We developed a single-stranded, linear amplification based library preparation method to improve difficult sample situations such as low input amount and low qualities, and achieved high single site sensitivity at the 0.1% allele frequency. Such features imply SLA-Seq has important clinical significance in the ctDNA/MRD detection.

Enhanced ctDNA Profiling Reveals Molecular Determinants of Response and Resistance in Relapsed and Refractory Mantle Cell Lymphoma (NLG‐MCL7‐VALERIA)

Introduction: Deciphering molecular determinants of response to targeted treatments and early detection of refractoriness in patients with relapsed and refractory (R/R) mantle cell lymphoma (MCL) could individualize treatment, improve outcomes and limit unnecessary toxicities. Circulating tumor DNA (ctDNA) allows non-invasive profiling and measurement of treatment responses. Yet, increased sensitivity for detection and distinction from confounding biological signals are warranted. Enhanced ctDNA detection can be achieved using duplex sequencing or tracing of phased variants. Poor recovery of complementary strands and low number of phased variants limit the feasibility of these approaches separately in MCL. Methods: Thus, we developed and optimized a hybrid ctDNA sequencing platform, which combines strand- and phasing-aware error correction methods for improved specificity and sensitivity of non-invasive B-cell lymphoma profiling, quantification and detection (Figure 1A). We applied this platform to profile ctDNA in serial plasma samples from 59 patients with R/R MCL treated with lenalidomide, venetoclax and rituximab in the Nordic phase II trial (NLG-MCL7; VALERIA). We compared the results with outcomes and real-time quantified PCR (RQ-PCR, ‘Euro-Minimal Residual Disease (MRD’)) data from bone marrow (BM) and peripheral blood (PB) samples that guided therapy in the trial (Figure 1B). Results: Joint analysis of targeted sequences from pretreatment tumor, PB, BM, and plasma samples revealed MCL genotypes for ctDNA tracing even in the patients who failed primer designs for Euro-MRD (Figure 1C). Comparison between genotyping of samples from different compartments from individual patients mitigated confounding clonal hematopoiesis background and revealed spatially-confined MCL mutations. TP53 mutated MCL conferred resistance to therapy, whereas SMARCA4 mutations translated into durable molecular remission (Figure 1D-E). Elevated pretreatment ctDNA concentration was associated with multiple clinical risk factors, leukemic disease, and poor outcome (Figure 1F). After six months of therapy, ctDNA levels dropped in all studied patients. The responses in the ctDNA were highly concordant with concomitant Euro-MRD and extended to patients not evaluable by RQ-PCR. Notably, contrasting ctDNA clearance, the analysis of the post-therapy cell-free DNA revealed positive selection of clonal hematopoiesis drivers following therapy exposure, such as TP53 mutations in 77% of the patients. The research was funded by: Academy of Finland, iCAN Flagship, University of Helsinki, Helsinki University Hospital, Finnish Cancer Organisation, Sigrid Juselius Foundation and Abbvie. Keywords: diagnostic and prognostic biomarkers, liquid biopsy, molecular targeted therapies No conflicts of interests pertinent to the abstract.

Results of phase I-II bridging study for Nous-209, a neoantigen cancer immunotherapy, in combination with pembrolizumab as first line treatment in patients with advanced dMMR/MSI-h colorectal cancer.

e14665 Background: Microsatellite instability (MSI) is a molecular hallmark in which systemic deficiency of DNA mismatch-repair (dMMR) generates tumor-specific frameshift peptides (FSPs). Nous-209 is an off-the-shelf cancer vaccine targeting 209 FSPs selected from dMMR/MSI-H tumors (Leoni, G. et al., 2020). Treatment options for unresectable dMMR/MSI-H tumors have improved, but further efforts to increase long-term progression-free survival are needed. NOUS-209-01 Phase I demonstrated that Nous-209 and pembrolizumab is safe, well tolerated, and provides clinical benefit in advanced gastric and CRC dMMR/MSI-H tumors (Fakih et al., 2022). Moreover, Nous-209 induces durable specific anti-tumoral immune response, capable of successfully infiltrating the tumor (D’Alise et al., 2022). Here, we present data from NOUS-209-01 study Phase I-II expansion cohort at the recommended phase 2 dose (RP2D) of Nous-209 and pembrolizumab in adults with dMMR/MSI-H locally advanced unresectable or metastatic colorectal cancer (mCRC) eligible for 1st line immune checkpoint inhibition. Methods: NOUS-209-01 ( NCT04041310 ) Phase I-II bridge expansion cohort receives one GAd20-209-FSP prime intramuscularly at the 1st infusion of pembrolizumab, followed by 3 MVA-209-FSP boosts with the 2nd, 3rd and 4th pembrolizumab cycles. Patients can continue treatment with pembrolizumab for 18 months. Response assessment is evaluated every 9 (+/-1) weeks by CT imaging, according to RECIST v1.1 criteria. ctDNA assessments are at baseline, 11, 28, 49, 79 weeks and at objective progression on study. Results: Phase I-II bridge enrolled 8 patients with dMMR/MSI-H treatment-naïve mCRC. One patient had clinical progression before 1st scan and was excluded as non-evaluable and 7 were evaluable for tumor response. 5/7 (71%, CI: 95% 29.0, 96.3) patients achieved objective response: 1 (14%) complete response (CR) and 4 (57%) partial responses (PR). 2 patients (29%) had disease progression (PD) at the 1st scan. ctDNA assessments were available for 6 out of 7 patients (4 PR; 2 PD); ctDNA levels reduced >50% at week 11 in the 4 PR patients, in contrast to a 28% increase in 1 PD. Interestingly 1 PD patient had ctDNA > 50% reduction. The median follow-up at data cut-off was 7.5 months (range: 3.3-8.4), median progression free survival and median duration of response have not been reached. 6/7 (85.7%) patients had treatment emergent adverse events (TEAE), all mild or moderate grade; no TEAEs were serious or led to treatment discontinuation. Conclusions: The combination of Nous-209 and pembrolizumab is safe, well tolerated and shows encouraging clinical efficacy in patients with treatment-naive dMMR/MSI-H mCRC eligible for anti-PD-1 therapy. The study is ongoing and expanding to Phase II randomization with a new accelerated Nous-209 vaccination schedule. Clinical trial information: NCT04041310 .

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: While nearly all younger patients (pts) with Hodgkin's lymphoma (HL) are cured with conventional chemotherapy, outcomes are significantly worse in those over age 60. Intriguingly, among older HL pts, those with EBV+ disease have even poorer outcomes (Keegan et al., JCO 2005), yet the biological basis for this phenomenon is not understood. The paucity of malignant cells in HL tumors has hampered genomic studies. Circulating tumor DNA (ctDNA) profiling can overcome these limitations, allowing noninvasive genotyping and risk stratification in HL (Spina et al., Blood 2018; Alig et al., ASH 2022). Here, we use a fully non-invasive approach for genomic profiling of elderly HL pts, focusing on the impact of ctDNA and EBV status on clinical outcomes. Methods: We profiled 57 pts (64%) from NCT02414568, a prospective, multicenter phase II LYSA-PVAB study of newly diagnosed classical HL >60 years (Ghesquières et al., ASH2019). All pts received 6 cycles of PVAB (prednisone, vinblastine, doxorubicin, and bendamustine). Plasma cfDNA was genotyped using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq); MRD was monitored using Phased variant Enrichment and Detection Sequencing (PhasED-Seq). Pts were classified into high and low pretreatment ctDNA levels using a threshold of 2.5 log hGE/mL (Alig et al., ASH2022). We defined EBV status either by LMP1 staining of FFPE tumor tissue or by VirCAPP-Seq targeting 180 viral species, where a predefined threshold of 32 genomes/ml was previously validated as 94% accurate (Garofalo et al., ASH 2022). Results: Pretreatment ctDNA was detected in 94% of pts with a median concentration of 2.2% (MAF), which was significantly correlated with disease burden measured by TMTV (p = 0.034). Pts with high pre-treatment ctDNA levels (39%) had significantly worse PFS (HR = 2.3, p = 0.039). EBV+ pts also had inferior PFS (HR = 2.2, p = 0.04) and OS (HR = 3.1, p = 0.028) (Figures A-B). In multivariate analysis, both indices remained independently associated with worse outcomes (HR = 2.5, p = 0.01 for pre-treatment ctDNA and HR = 2.7, p = 0.01 for EBV status). Interestingly, pre-treatment ctDNA levels did not differ between EBV+ and EBV- tumors (p = 0.92), while specific genetic aberrations were significantly associated with respective EBV status (Figure C). Among evaluable pts for MRD monitoring, detection of ctDNA after four cycles of chemotherapy was highly associated with risk of death (Figure D). The research was funded by: NIH/NCI R01 R01CA257655 Keywords: Diagnostic and Prognostic Biomarkers, Genomics, Epigenomics, and Other -Omics, Hodgkin lymphoma Conflicts of interests pertinent to the abstract. C. Rossi Consultant or advisory role: Genmab, Janssen, Incyte Educational grants: Kyte, Abbvie S. K. Alig Consultant or advisory role: Takeda Pharmaceuticals D. M. Kurtz Consultant or advisory role: Adaptive Biotechnologies, Roche, Foresight O. Casasnovas Consultant or advisory role: Roche, Takeda Educational grants: Roche, Takeda, BMS, Janssen A. A. Alizadeh Consultant or advisory role: Adaptive Biotechnologies, Foresight Diagnostics, Roche, BMS, Gilead, Genentech, Karyopharm

Early ctDNA reduction may identify patients with stable disease and long OS on tebentafusp.

9589 Background: Tebentafusp, a bispecific (gp100 × CD3) ImmTAC showed significant overall survival (OS) benefit (HR 0.51) versus investigator’s choice in a Phase (Ph) 3 trial in first line (1L) HLA-A*02:01+ adult patients (pts) with metastatic uveal melanoma (mUM) [Nathan NEJM 2021]. In Ph2 and Ph3 trials of tebentafusp, OS was improved regardless of RECIST best response and the degree of early reduction in ctDNA was a better predictor of OS [Carvajal Nat Med 2022; Sullivan AACR 2023]. Here we explored whether early on-treatment reduction in ctDNA could distinguish pts with stable disease (SD) and long OS vs those with SD and short OS in a Ph3 trial of tebentafusp in 1L mUM pts. Methods: 1L HLA-A*02:01+ pts with mUM received 68 mcg tebentafusp weekly intravenously after intra-patient dose escalation (NCT03070392). Response was assessed by investigators per RECISTv1.1. Sera collected at baseline (BL) and week 9 on tebentafusp were analyzed for ctDNA using targeted mPCR-NGS assay for mutations in 15 genes including GNAQ, GNA11, SF3B1, CYSLTR2, PLCB4 and EIF1AX. Landmark OS from week 9 was analyzed in subsets with ≥0-3 log reductions in mean tumor molecules (MTM) per ml of serum on treatment. Data cutoff 11-Nov-2022. Results: 76/245 (36%) tebentafusp-treated pts had best response of SD, with a median OS of 29 months and were evaluable for ctDNA. Baseline tumor burden as assessed by sum of longest lesion diameters and percentage of pts with elevated BL serum LDH were greater in the subset with < 1 year (yr) OS (median 70 mm and 64%, respectively) than in subsets with OS ≥ 1 yr (median 42 mm, 11%) or OS ≥ 2 yrs (median 31 mm, 9%). Almost half (36/76; 47%) of all SD pts had detectable ctDNA mutations in one or more UM genes at baseline. Baseline ctDNA levels were similar across survival groups. By week 9 on-treatment, ctDNA reduction was observed in 34/36 (94%) evaluable SD pts, including 29/36 (81%) with ≥ 0.5 log reduction, 18/36 (50%) with ≥ 2 log reduction and 16/36 (44%) with undetectable ctDNA (clearance). Deeper reductions in ctDNA were associated with better OS (Table). Conclusions: In this Ph3 trial, ctDNA reduction by week 9 on tebentafusp was strongly associated with improved OS in pts with best RECIST SD. Early ctDNA reduction may predict SD patients with long OS on tebentafusp. [Table: see text]

Accuracy of genome-wide mutational analysis for tumor-informed ctDNA-guided MRD monitoring in bladder cancer.

4589 Background: Neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is the standard management of localized muscle-invasive bladder cancer (MIBC). However, about 45% of patients develop metastases within 2 years after RC. Biomarkers for treatment efficacy evaluation and early detection of minimal residual disease (MRD) are needed for earlier treatment initiation and monitoring of treatment response. Tumor-informed detection of mutations in cell-free DNA (cfDNA) has shown promising results to monitor MRD. However, the low tumor fraction after surgery and limited input material obtained from a typical plasma sample limits the probability of detecting low metastatic burden scenarios. Here we implemented and applied a whole-genome sequencing (WGS) approach to monitor circulating tumor DNA (ctDNA) for sensitive ctDNA detection. Methods: 110 MIBC patients undergoing NAC and RC were enrolled. cfDNA was extracted from ~1mL plasma (n=978) and procured from longitudinal plasma sampling during NAC and pre-RC and post-RC. WGS of tumor/germline pairs (30x/20x) and plasma cfDNA (>20x) was performed, facilitating detection of genome-wide genomic alterations and quantification of ctDNA using the C2inform method. Results: For each patient we developed a tumor-informed WGS model by integrating genome-wide mutation and copy number variation data coupled with advanced signal processing and AI-based error suppression. Patient-specific somatic variant patterns were used for detection and measuring ctDNA levels in low-input blood samples by WGS. Post-RC ctDNA analysis identified patients with recurrence with 78% sensitivity and 95% specificity and with a median lead time over radiographic imaging of 118 days (full follow-up included). When restricting follow-up time to 12 months following the latest ctDNA test, a sensitivity of 86% and specificity of 94% was achieved. ctDNA status was associated with recurrence-free (p<0.0001) and overall survival (p<0.0001). Furthermore, ctDNA clearance during NAC was also associated with recurrence-free survival (p=0.0051). The APOBEC associated signatures SBS2 and SBS13 were identified as the primary contributors to the mutational landscape of the primary tumors and TP53, KMT2D, PIK3CA, RB1 and KDM6A were the main affected driver genes. Half of the tumors displayed whole genome doubling. Moreover, our analysis of plasma samples after treatment showed the presence of chemotherapy-induced mutational signatures not present in the primary tumor, along with increases in copy numbers on chromosomes 19q and 20, and a focal amplification of the FGFR3 gene on chromosome 4. Conclusions: Our results highlight the clinical potential of personalized genome-wide mutation integration as an ultra-sensitive, non-invasive method for MRD detection and treatment response monitoring.

Clinical outcomes and correlation with longitudinal circulating tumor (ct)DNA dynamics of a phase I/II study of GSK525762 combined with fulvestrant in patients with hormone receptor-positive/HER2-negative (HR+/HER2−) advanced or metastatic breast cancer.

1073 Background: Endocrine therapy is the main treatment option for hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+/HER2−) metastatic breast cancer (mBC). However, many patients experience disease progression due to resistance to endocrine therapy. Molibresib (GSK525762) is a small-molecule inhibitor of bromodomain and extra-terminal (BET) family proteins (BRD2, BRD3, BRD4, and BRDT). Pre-clinical data suggested that the combination of molibresib with endocrine therapy might overcome endocrine resistance. This study aimed to investigate the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy (objective response rate [ORR]) of molibresib combined with fulvestrant in women with HR+/HER2− mBC. The association between early ctDNA dynamics and clinical outcomes was also assessed. Methods: In this phase I/II dose-escalation and expansion study, patients received oral molibresib 60 mg or 80 mg once daily in combination with intramuscular fulvestrant. Patients enrolled had relapsed/refractory, advanced/metastatic HR+/HER2− BC with disease progression on prior treatment with an aromatase inhibitor, with or without a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Baseline and week 4 plasma samples were collected and tested using a 74-gene ctDNA panel. Molecular response (MR) was defined as at least 50% reduction of baseline ctDNA level calculated as the average allele frequency of detected single nucleotide variants or indels. Results: The study included 123 patients. The 3 most common molibresib-related adverse events (AEs) were nausea (52%), dysgeusia (49%), and fatigue (44%). At molibresib 60 mg, >90% patients experienced treatment-related AEs, with varying incidence of Grade ≥3 AEs (17%–71%) amongst subgroups. The ORR was 13% (95% confidence interval [CI], 8–20), not meeting the 25% threshold for proceeding to phase II. Among 82 patients with detected baseline circulating tumor DNA, a strong association was observed between baseline copy number amplification (CNA) presence and poor progression-free survival (PFS) with hazard ratio (HR) of 2.89 (95% CI, 1.73–4.83; P < 0.0001). MR was significantly correlated with better PFS (HR=0.38; 95% CI, 0.19–0.75; P = 0.0037), which was further improved when MR was refined as no baseline CNA (HR=0.22; 95% CI, 0.09–0.54; P = 0.0003). Conclusions: Molibresib in combination with fulvestrant did not demonstrate clinically meaningful activity in this study. Copy number adjusted ctDNA MR is a promising early marker for clinical benefit. Clinical trial information: NCT02964507 .

Polatuzumab vedotin with infusional chemotherapy for untreated aggressive B-cell non-Hodgkin lymphomas.

The POLARIX trial demonstrated the superiority of polatuzumab vedotin (Pola) over vincristine in the rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) regimen for large B-cell lymphomas, but it is unknown whether Pola can be safely incorporated into intensified regimens (eg, dose-adjusted [DA]-EPOCH-R [etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab]) typically used for the highest risk histologies. This was a single-center, open-label, prospective clinical trial of 6 cycles of Pola-DA-EPCH-R (vincristine omitted) in aggressive large B-cell lymphomas. The primary end point was to estimate the safety of Pola-DA-EPCH-R as measured by the rate of dose-limiting toxicities (DLTs) in the first 2 cycles with prespecified suspension rules. Secondary and exploratory end points included efficacy and correlation with circulating tumor DNA (ctDNA) levels. We enrolled 18 patients on study, and with only 3 DLTs observed, the study met its primary end point for safety. There were 5 serious adverse events, including grade 3 febrile neutropenia (3, 17%), grade 3 colonic perforation in the setting of diverticulitis, and grade 5 sepsis/typhlitis. Among 17 evaluable patients, the best overall response rate was 100%, and the complete response rate was 76%. With a median follow-up of 12.9 months, 12-month event-free survival was 72%, and 12-month overall survival was 94%. No patient with undetectable ctDNA at the end of treatment has relapsed to date. Using Pola to replace vincristine in the DA-EPOCH-R regimen met its primary safety end point. These data support the further evaluation and use of this approach in histologies where the potential benefit of both an intensified regimen and Pola may be desired. This trial was registered at www.clinicaltrials.gov as #NCT04231877.

Molecular risk factors for locoregional recurrence in resected non-small cell lung cancer.

Locoregional recurrence is of high risk and is associated with a poor prognosis in terms of OS for non-small cell lung cancer (NSCLC). Local control is essential for radical cure of NSCLC. Previous studies have investigated the clinicopathological risk factors for locoregional recurrence, but the genomic biomarkers associated with locoregional recurrence have been inadequately studied. A total of 118 patients who underwent tumor resection with mutation-detected tumor specimens were included. Tumor samples at surgery and pretreatment/postoperative blood samples were collected for mutational profiling. Among 48 patients with disease recurrence, 46% developed locoregional recurrence (LR) and 75% developed distant metastasis (DM). The 3-year actuarial risk of LR and DM was 25% and 43%, respectively. The first sites of failure were locoregional only (29%), locoregional and distant (10%), and distant only (61%). Patients with LR showed significantly higher ctDNA level than those with only DM at the time of initial recurrence. On multivariate analysis of baseline risk factors, the presence of allele frequency heterogeneity and baseline ctDNA shedding were found to be independently associated with a higher risk of LR. Patients with disruptive TP53 mutations had significantly lower LR-free survival as compared to patients with wild-type TP53 or nondisruptive mutations. EGFR mutations showed a favorable prognostic value for LR and is not induced by EGFR tyrosine kinase inhibitor therapy. Both disruptive TP53 mutation and EGFR mutation remained the significant prognostic factor after adjustment for histological type, pathologic nodal stage and adjuvant therapy. Nearly half of disease recurrences after surgery for NSCC involved locoregional sites. We identified genomic biomarkers from baseline tumor and ctDNA samples which showed promising prognostic value for LR only. This can help identify patients who had a higher risk of locoregional recurrence regardless of the risk of distant metastasis.

Early serial circulating tumor DNA sequencing predicts the efficacy of chemohormonal therapy in patients with metastatic hormone-sensitive prostate cancer

Chemohormonal therapy is a standard treatment for metastatic hormone-sensitive prostate cancer (mHSPC); however, there are no biomarkers to guide clinical decisions regarding therapeutic options. We aimed to evaluate the clinical utility of serial circulating tumor DNA (ctDNA) sequencing in early prediction of the efficacy of chemohormonal therapy in patients with mHSPC. We conducted a retrospective observational study of 66 patients with mHSPC receiving chemohormonal therapy who underwent serial targeted gene-panel ctDNA sequencing. Peripheral blood samples were collected before treatment and after one cycle of chemotherapy. Kaplan–Meier and log-rank analyses were used to analyze the association between ctDNA status and disease progression-free survival. Serial changes in the ctDNA fraction and genetic alterations were also observed. After one cycle of chemotherapy, 23 (34.8%) patients displayed elevated ctDNA levels, whereas the other patients (65.2%, n = 43) did not. The median time to castration resistance in the group with reduced ctDNA levels was significantly longer than that in the group with increased ctDNA levels (17.70 vs. 8.43 months [mo], p < 0.001). Interestingly, patients with de novo alterations in homologous recombination pathway genes after treatment experienced a shorter time to castration resistance than that experienced by the remaining patients (8.02 vs. 13.20 mo, p = 0.011). The increased ctDNA levels or de novo alterations detected in homologous recombination pathway genes are a harbinger of disease progression. Early serial ctDNA sequencing could aid clinicians in making accurate treatment decisions.

Plasma ESR1 mutations and outcome to first-line paclitaxel and bevacizumab in patients with advanced ER-positive/HER2-negative breast cancer

BackgroundESR1 mutations have been identified as mechanism for endocrine resistance and are also associated with a decreased overall survival. We assessed ESR1 mutations in circulating tumor DNA (ctDNA) for impact on outcome to taxane-based chemotherapy in advanced breast cancer patients.MethodsESR1 mutations were determined in archived plasma samples from patients treated with paclitaxel and bevacizumab (AT arm, N = 91) in the randomized phase II ATX study. Samples collected at baseline (n = 51) and at cycle 2 (n = 13, C2) were analyzed using a breast cancer next-generation sequencing panel. This study was powered to detect a benefit in progression-free survival (PFS) at six months for patients treated with paclitaxel/bevacizumab compared to historical trials with fulvestrant. PFS, overall survival (OS), and ctDNA dynamics were exploratory analyses.ResultsPFS at six months was 86% (18/21) in patients with an ESR1 mutation detected and 85% (23/27) in wildtype ESR1 patients. In our exploratory analysis, median progression-free survival (PFS) was 8.2 months [95% CI, 7.6–8.8] for ESR1 mutant patients versus 8.7 months [95% confidence interval (CI), 8.3–9.2] for ESR1 wildtype patients [p = 0.47]. The median overall survival (OS) was 20.7 months [95% CI, 6.6–33.7] for ESR1 mutant patients versus 28.1 months [95% confidence interval (CI), 19.3–36.9] for ESR1 wildtype patients [p = 0.27]. Patients with ≥ two ESR1 mutations had a significantly worse OS, but not PFS, compared to those who did not [p = 0.003]. Change in ctDNA level at C2 was not different between ESR1 and other mutations.ConclusionsPresence of ESR1 mutations in baseline ctDNA might not be associated with inferior PFS and OS in advanced breast cancer patients treated with paclitaxel/bevacizumab.

Monitoring Treatment Response, Early Recurrence, and Survival in Uterine Serous Carcinoma and Carcinosarcoma Patients Using Personalized Circulating Tumor DNA Biomarkers.

Uterine serous carcinoma (USC) and carcinosarcomas (CSs) are rare, highly aggressive variants of endometrial cancer. No reliable tumor biomarkers are currently available to guide response to treatment or detection of early recurrence in USC/CS patients. Circulating tumor DNA (ctDNA) identified using ultrasensitive technology such as droplet digital polymerase chain reaction (ddPCR) may represent a novel platform for the identification of occult disease. We explored the use of personalized ctDNA markers for monitoring USC and CS patients. Tumor and plasma samples from USC/CS patients were collected at the time of surgery and/or during the treatment course for assessment of tumor-specific somatic structural variants (SSVs) by a clinical-grade next-generation sequencing (NGS) platform (i.e., Foundation Medicine) and a droplet digital PCR instrument (Raindance, ddPCR). The level of ctDNA was quantified by droplet digital PCR in plasma samples and correlated to clinical findings, including CA-125 serum and/or computed tomography (CT) scanning results. The genomic-profiling-based assay identified mutated "driver" target genes for ctDNA analysis in all USC/CS patients. In multiple patients, longitudinal ctDNA testing was able to detect the presence of cancer cells before the recurrent tumor was clinically detectable by either CA-125 or CT scanning. Persistent undetectable levels of ctDNA following initial treatment were associated with prolonged progression-free and overall survival. In a USC patient, CA-125 and TP53 mutations but not PIK3CA mutations become undetectable in the plasma at the time of recurrence, suggesting that more than one customized probe should be used for monitoring ctDNA. Longitudinal ctDNA testing using tumor-informed assays may identify the presence of residual tumors, predict responses to treatment, and identify early recurrences in USC/CS patients. Recognition of disease persistence and/or recurrence through ctDNA surveillance may allow earlier treatment of recurrent disease and has the potential to change clinical practice in the management of USC and CS patients. CtDNA validation studies in USC/CS patients prospectively enrolled in treatment trials are warranted.

Personalized circulating tumor DNA detection to monitor immunotherapy efficacy and predict outcome in locally advanced or metastatic non-small cell lung cancer.

Immune checkpoint inhibitors (ICIs) or combined with chemotherapy exhibit substantial efficacy for the treatment of advanced non-small cell lung cancer (NSCLC). However, reliable biomarkers that can monitor response to first-line ICIs ± chemotherapy remain unclear. A total of 16 tumor tissues and 46 matched peripheral blood samples at baseline and during treatment were retrospectively collected from 19 locally advanced or metastatic NSCLC patients. The circulating tumor DNA (ctDNA) burden by tumor-informed assay was detected to monitor and predict the therapeutic response and survival of NSCLC patients treated with first-line ICIs or plus chemotherapy. We found that ctDNA was only positively detected in one patient by tumor-agnostic assay with a mean variant allele fraction (VAF) of 6.40%, whereas it was positively detected in three patients by tumor-informed assay with a mean VAF of 8.83%, 0.154%, and 0.176%, respectively. Tumor-informed assays could sensitively detect ctDNA in 93.75% (15/16) of patients. Trends in the level of ctDNA from baseline to first evaluation was consistent with the radiographic changes. There was a greater decrease in ctDNA after treatment compared with baseline in patients with partial response compared to patients with stable disease/progressive disease. Patients with over a 50% reduction in ctDNA had a significant progression-free survival and overall survival benefit. The tumor-informed assay was favorable for ctDNA detection, and early dynamic changes in plasma ctDNA may be a valuable biomarker for monitoring the efficacy and predicting the outcome in advanced NSCLC patients treated with first-line ICIs ± chemotherapy.

NK cell activity and methylated HOXA9 ctDNA as prognostic biomarkers in patients with non-small cell lung cancer treated with PD-1/PD-L1 inhibitors

BackgroundPD-1/PD-L1 inhibitors have improved survival for patients with non-small cell lung cancer (NSCLC). We evaluated natural killer cell activity (NKA) and methylated HOXA9 circulating tumor DNA (ctDNA) as prognostic biomarkers in NSCLC patients treated with PD-1/PD-L1 inhibitors.MethodsPlasma was prospectively collected from 71 NSCLC patients before treatment with PD-1/PD-L1 inhibitors and before cycles 2–4. We used the NK Vue® assay to measure the level of interferon gamma (IFNγ) as a surrogate for NKA. Methylated HOXA9 was measured by droplet digital PCR.ResultsA score combining NKA and ctDNA status measured after one treatment cycle had a strong prognostic impact. Group 1 had IFNγ < 250 pg/ml and detectable ctDNA (n = 27), group 2 consisted of patients with either low levels of IFNγ and undetectable ctDNA or high levels of IFNγ and detectable ctDNA (n = 29), group 3 had IFNγ ≥250 pg/ml and undetectable ctDNA (n = 15). Median OS was 221 days (95% CI 121–539 days), 419 days (95% CI 235–650 days), and 1158 days (95% CI 250 days—not reached), respectively (P = 0.002). Group 1 had a poor prognosis with a hazard ratio of 5.560 (95% CI 2.359–13.101, n = 71, P < 0.001) adjusting for PD-L1 status, histology, and performance status.ConclusionsCombining NKA and ctDNA status after one cycle of treatment was prognostic in patients with NSCLC treated with PD-1/PD-L1 inhibitors.

Mutation profiling, tumour burden assessment, outcome prediction and disease monitoring by circulating tumour DNA in peripheral T-cell lymphoma.

In this prospective study, we aimed to evaluate the utility of circulating tumour DNA (ctDNA) in peripheral T-cell lymphomas (PTCLs). Plasma cell-free DNA (cfDNA) was obtained, and the mutational profile was assessed in 47 patients with newly diagnosed mature T- and NK-cell lymphoma. To validate the mutations detected in cfDNA, paired tumour tissue samples were available for 36 patients. Targeted next-generation sequencing was performed. A total of 279 somatic mutations involving 149 genes were identified in the 47 cfDNA samples. The overall sensitivity of plasma cfDNA in discovering biopsy-confirmed mutations was 73.9% with a specificity of 99.6%. When we considered only mutations with variant allele frequency >5% in the tumour biopsy, the sensitivity increased to 81.9%. Pretreatment ctDNA concentration and the number of mutations were highly correlated with tumour burden indicators including lactate dehydrogenase, Ann Arbor stage and International Prognostic Index score. Patients with high ctDNA level (>1.9 log ng/mL) had significantly lower overall response rates, inferior 1-year progression-free survival and overall survival rates than those with low level. Longitudinal analysis of ctDNA showed a strong agreement between ctDNA dynamics and the radiographic response. In conclusion, our study demonstrates that ctDNA may serve as a promising tool for mutational profiling, tumour burden assessment, outcome prediction and disease monitoring in PTCLs.

Genomic alterations of cerebrospinal fluid cell-free DNA in leptomeningeal metastases of gastric cancer

BackgroundLeptomeningeal metastases (LM) were rare in gastric cancer (GC), and GC patients with LM (GCLM) generally suffer from poor prognosis. Nevertheless, the clinical utility of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) was underinvestigated in GCLM.MethodsWe retrospectively studied 15 GCLM patients, and all patients had paired primary tumor tissue samples and post-LM CSF samples while 5 patients also had post-LM plasma samples. All samples were analyzed using next-generation sequencing (NGS), and the molecular and clinical features were correlated with clinical outcomes.ResultsCSF had higher mutation allele frequency (P = 0.015), more somatic mutations (P = 0.032), and more copy-number variations (P < 0.001) than tumor or plasma samples. Multiple genetic alterations and aberrant signal pathways were enriched in post-LM CSF, including CCNE1 amplification and cell cycle-related genes, and CCNE1 amplification was significantly associated with patients’ overall survival (P = 0.0062). More potential LM progression-related markers were detected in CSF samples than in tumor samples, including PREX2 mutation (P = 0.014), IGF1R mutation (P = 0.034), AR mutation (P = 0.038), SMARCB1 deletion (P < 0.001), SMAD4 deletion (P = 0.0034), and TGF-beta pathway aberration (P = 0.0038). Additionally, improvement in intracranial pressure (P < 0.001), improvement in CSF cytology (P = 0.0038), and relatively low levels of CSF ctDNA (P = 0.0098) were significantly associated with better PFS. Lastly, we reported a GCLM case whose CSF ctDNA dynamic changes were well correlated with his clinical assessment.ConclusionsCSF ctDNA could more sensitively detect molecular markers and metastasis-related mechanisms than tumor tissues in GCLM patients, and our study sheds light on utilizing CSF ctDNA in prognostic estimation and clinical assessment in GCLM.

Blood tumor mutational burden and dynamic changes in circulating tumor DNA predict response to pembrolizumab treatment in advanced non-small cell lung cancer.

The use of immunotherapy targeting the programmed cell death protein-1 (PD-1) and its ligand (PD-L1) has provided new hope for patients with non-small cell lung cancer (NSCLC). However, good biomarkers are needed to identify which patients will benefit from the treatment. In this study, we investigated if circulating tumor DNA (ctDNA) could predict response to pembrolizumab. Plasma samples from patients with NSCLC treated with pembrolizumab were collected immediately before and after one or two cycles of treatment. ctDNA was isolated and analyzed using targeted next-generation sequencing with a lung cancer gene panel. Mutations were detected in ctDNA in 83.93% of patients before treatment initiation. High blood tumor mutational burden (bTMB), measured as the number of different mutations per Mb panel, correlated to longer progression-free survival (PFS) (10.45 vs. 2.30 months) and overall survival (OS) (21.80 vs. 12.20 months), whereas no predictive value was found in the number of mutant molecules per mL of plasma. The absence of mutations just after treatment initiation correlated with improved PFS (20.25 vs. 4.18 months) and OS (28.93 vs. 15.33 months). High bTMB before treatment was associated with a decreasing ctDNA level after treatment initiation. Importantly, a subgroup of patients experienced an increase in the ctDNA level after treatment initiation, and this correlated with inferior PFS (2.19 vs. 11.21 months) and OS (7.76 vs. 24.20 months). All patients in the subgroup with increased ctDNA level progressed within 10 months. Monitoring of ctDNA contains vital information about response to therapy, where the bTMB and the dynamics in the initial part of treatment are particularly important for response. Increasing ctDNA levels after treatment initiation are significantly correlated with inferior survival.