Mutations provide the raw material of evolution, and thus our ability to study evolution depends fundamentally on having precise measurements of mutational rates and patterns. We generate a data set ...for this purpose using (1) de novo mutations from mutation accumulation experiments and (2) extremely rare polymorphisms from natural populations. The first, mutation accumulation (MA) lines are the product of maintaining flies in tiny populations for many generations, therefore rendering natural selection ineffective and allowing new mutations to accrue in the genome. The second, rare genetic variation from natural populations allows the study of mutation because extremely rare polymorphisms are relatively unaffected by the filter of natural selection. We use both methods in
, first generating our own novel data set of sequenced MA lines and performing a meta-analysis of all published MA mutations (∼2000 events) and then identifying a high quality set of ∼70,000 extremely rare (≤0.1%) polymorphisms that are fully validated with resequencing. We use these data sets to precisely measure mutational rates and patterns. Highlights of our results include: a high rate of multinucleotide mutation events at both short (∼5 bp) and long (∼1 kb) genomic distances, showing that mutation drives GC content lower in already GC-poor regions, and using our precise context-dependent mutation rates to predict long-term evolutionary patterns at synonymous sites. We also show that de novo mutations from independent MA experiments display similar patterns of single nucleotide mutation and well match the patterns of mutation found in natural populations.
Neoadjuvant immunotherapies hold promise in muscle-invasive bladder cancer (MIBC).
To report on 2-yr disease-free (DFS) and overall (OS) survival including novel tissue-based biomarkers and ...circulating tumor DNA (ctDNA) in the ABACUS trial.
ABACUS was a multicenter, single-arm, neoadjuvant, phase 2 trial, including patients with MIBC (T2-4aN0M0) who were ineligible for or refused neoadjuvant cisplatin-based chemotherapy.
Two cycles of atezolizumab were given prior to radical cystectomy. Serial tissue and blood samples were collected.
The primary endpoints of pathological complete response (pCR) rate and dynamic changes to T-cell biomarkers were published previously. Secondary outcomes were 2-yr DFS and OS. A biomarker analysis correlated with relapse-free survival (RFS) was performed, which includes FOXP3, major histocompatibility complex class I, CD8/CD39, and sequential ctDNA measurements.
The median follow-up time was 25 mo (95% confidence interval CI 25-26). Ninety-five patients received at least one cycle of atezolizumab. Eight patients did not undergo cystectomy (only one due to disease progression). The pCR rate was 31% (27/88; 95% CI 21-41). Two-year DFS and OS were 68% (95% CI 58-76) and 77% (95% CI 68-85), respectively. Two-year DFS in patients achieving a pCR was 85% (95% CI 65-94). Baseline PD-L1 and tumor mutational burden did not correlate with RFS (hazard ratio HR 0.60 95% CI 0.24-1.5, p = 0.26, and 0.72 95% CI 0.31-1.7, p = 0.46, respectively). RFS correlated with high baseline stromal CD8+ (HR 0.25 95% CI 0.09-0.68, p = 0.007) and high post-treatment fibroblast activation protein (HR 4.1 95% CI 1.3-13, p = 0.01). Circulating tumor DNA positivity values at baseline, after neoadjuvant therapy, and after surgery were 63% (25/40), 47% (14/30), and 14% (five/36), respectively. The ctDNA status was highly prognostic at all time points. No relapses were observed in ctDNA-negative patients at baseline and after neoadjuvant therapy. The lack of randomization and exploratory nature of the biomarker analysis are limitations of this work.
Neoadjuvant atezolizumab in MIBC is associated with clinical responses and high DFS. CD8+ expression and serial ctDNA levels correlated with outcomes, and may contribute to personalized therapy in the future.
We showed that bladder cancer patients receiving immunotherapy followed by cystectomy have good long-term outcomes. Furthermore, we found that certain biological features can predict patients who might have particular benefit from this therapy.
4506 Background: In IMmotion010, adj atezo did not prolong investigator-assessed disease-free survival (DFS; primary endpoint) vs pbo after resection in pts with RCC (HR: 0.93, 95% CI: 0.75, 1.15; ...P=0.50; Pal Lancet 2023). This exploratory analysis of circulating protein biomarkers was performed to identify high-risk pts with minimal residual disease (MRD) who may show differential benefit from treatment (tx) with atezo. Methods: Pts with RCC with clear-cell or sarcomatoid component and increased recurrence risk post nephrectomy were randomized 1:1 to atezo 1200 mg or pbo IV q3w for 16 cycles or 1 year. A retrospective proteomics analysis was done with an affinity-based proximity extension assay (PEA) panel of ≈3000 analytes to identify circulating proteins with differential abundance patterns in matched serum samples (baseline vs at recurrence). A high sensitivity electrochemiluminescence (ECL) assay was then used to evaluate levels of KIM-1, a membrane glycoprotein overexpressed in clear-cell and papillary RCC, in all available baseline and post-tx serum samples. Outcomes in pts with KIM-1 high (≥86 pg/mL) vs low status at baseline were analyzed. Results: In pts with matched PEA samples (n=73), circulating KIM-1 was identified as the most significantly enriched protein at recurrence vs baseline. Of 778 pts enrolled in IMmotion010, 752 (97%) had baseline KIM-1 data (high: 300 40%; low: 452 60%). KIM-1–high status was associated with reduced DFS, and pts with KIM-1 high had better DFS with atezo vs pbo (Table). Longitudinal analysis of matched samples showed that in KIM-1–high pts, 9% (12/138) and 26% (36/141) of pts had a ≥30% increase from baseline in KIM-1 levels at Cycle 4 Day 1 with atezo vs pbo, compared with 16% (34/213) and 12% (25/207) in KIM-1-low pts, respectively. A ≥30% KIM-1 increase was associated with worse DFS in both KIM-1–high (atezo HR: 1.68, 95% CI: 0.77, 3.69; pbo HR: 3.53, 95% CI: 2.24, 5.58) and KIM-1–low (atezo HR: 3.56, 95% CI 2.21, 5.75; pbo HR: 3.22, 95% CI: 1.81, 5.70) subgroups. In pts with matched ECL samples (n=103), median KIM-1 levels were higher ( P<0.001) at recurrence (172 pg/mL) than at baseline (79 pg/mL). Conclusions: In IMmotion010, high baseline serum KIM-1 levels were associated with poorer prognosis but improved clinical outcomes with atezo vs pbo. Increased post-tx KIM-1 was associated with worse DFS. These data suggest that circulating KIM-1 may be a non-invasive marker of MRD and disease recurrence and be associated with benefit from atezo in adj RCC. Further investigation of KIM-1 in adj RCC is warranted. Clinical trial information: NCT03024996 . Table: see text
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Background: IMbassador250 (IM250) was a prospective phase III trial which showed no overall survival (OS) benefit for adding atezolizumab to enzalutamide for men with mCRPC who had prior ...progression on abiraterone. Analyzing IM250, we previously demonstrated baseline TF and 75% reduction in TF (using a prototype TF) both to be prognostic. Here we hypothesized that early detection of and changes in ctDNA TF are associated with clinical responses. Methods: Pre-treatment (but post-abiraterone progression) plasma samples from IM250 were profiled using FoundationOne Liquid CDx (F1LCDx). To enable tumor-naïve ctDNA monitoring while avoiding non-tumor signal, we developed FoundationOne Monitor (F1M) which leverages the same sequencing platform as F1LCDx and enables quantification of and changes in ctDNA TF from prior F1LCDx or F1M results. Changes in status of TF (detected vs not detected) from baseline to cycle 3, day 1 (C3D1, 6 weeks), as well as detection at C3D1 alone, were compared vs radiographic progression-free survival (rPFS) and overall survival (OS), as well as changes in PSA (50% reduction). Results: A total of 418 patients with advanced mCRPC were included: median age: 70, median baseline PSA: 68.7 ng/ml, median baseline TF: 15.5%. In the cohort, 335 patients (80%) had detectable TF at baseline and 303 (72%) had detectable TF at C3D1. TF detected at C3D1 was associated with shorter rPFS (HR 3.24, 95% CI: 2.46-4.28, p<0.001) and OS (HR 5.03, 95% CI: 3.41-7.41, p<0.001). Patients with TF detected at both baseline and C3D1 had median rPFS: 4.1 months and median OS: 12.6 months. 88% of patients with “ctDNA detected” at C3D1 had a rPFS≤6 months. The positive predictive value (PPV) for ctDNA status in predicting non-durable response was 74% versus 67% for PSA. When TF and PSA reduction at C3D1 were discordant, patients with TF undetected/PSA not reduced had more favorable outcomes compared with PSA reduced/TF detected (median OS 22.1 months vs. 16 months, p<0.001). TF also provided additional stratification for patients with clinically ambiguous results who had no radiographic progression but lack of >50% PSA reduction, resulting in high and low risk populations (median OS 13.0 months vs 20.5 months, HR=3.80, p<0.001). Conclusions: Here we report on a new tumor-naïve monitoring assay (F1M) for molecular response assessment which is based on ctDNA TF detection and dynamics. TF detection at C3D1 was linked to unfavorable outcomes and identified early progression post-abiraterone, with increased information derived from TF detection at baseline. TF on F1M thus complements PSA testing, which had a lower PPV for identifying early progression than ctDNA. Together, we provide a non-invasive strategy that is independent of and additive to PSA and may refinepersonalized approaches tailored to the individual patient’s risk of progression. Clinical trial information: NCT03016312 .
Circulating-tumor DNA (ctDNA)-positive status was a negative prognostic biomarker in patients with muscle-invasive urothelial carcinoma after radical surgery and possibly a predictive biomarker for ...the overall survival benefit with adjuvant atezolizumab versus observation. The ctDNA-negative status possibly identified patients who experienced an unfavorable risk-benefit profile with adjuvant atezolizumab.
Interim results from IMvigor010 showed an overall survival (OS) benefit for adjuvant atezolizumab (anti–PD-L1) versus observation in patients with circulating tumor DNA (ctDNA)-positive muscle-invasive urothelial carcinoma (MIUC).
To report updated OS and safety by ctDNA status.
This ad hoc analysis from a global, open-label, randomized, phase 3 trial (NCT02450331) included intention-to-treat (ITT) population with evaluable cycle 1 day 1 (C1D1) ctDNA samples.
Atezolizumab (1200 mg every 3 wk) or observation for ≤1 yr.
OS, relapse rates, and safety by ctDNA status were assessed.
Among 581 of 809 ITT patients included, 214 (37%) were ctDNA positive. Atezolizumab did not improve OS versus observation in ITT patients (hazard ratio HR 0.91 95% confidence interval {CI} 0.73–1.13; median follow-up 46.8 mo interquartile range, 36.1–53.6). In the observation arm, ctDNA positivity versus negativity was associated with shorter OS (HR 6.3 95% CI 4.3–9.3). The ctDNA positivity identified patients with an OS benefit favoring atezolizumab versus observation (HR 0.59 95% CI 0.42–0.83). A greater reduction in ctDNA levels with atezolizumab (C3D1) was associated with longer OS (100% clearance, 60.0 mo 95% CI 35.5–not estimable; 50–99% reduction, 34.3 mo 95% CI 15.2–not estimable; <50% reduction, 19.9 mo 95% CI 16.4–32.2). The ctDNA positivity at C1D1 + C3D1 was associated with relapse with greater sensitivity than C1D1 alone (68% vs 57%). Adverse events were more frequent with atezolizumab than with observation, regardless of ctDNA status. A study limitation was its exploratory design.
Evidence suggests that ctDNA positivity in MIUC predicts a benefit with atezolizumab. An in-progress prospective study will further evaluate these findings.
Among patients with urothelial cancer after surgery, survival was poorer if tumor-derived DNA was detected in their bloodstream; these patients’ survival was longer with atezolizumab versus observation. Bloodstream tumor-derived DNA may identify patients who benefit from atezolizumab.
Abstract
Comprehensive genomic profiling (CGP) of circulating tumor DNA (ctDNA) provides an opportunity to noninvasively monitor a patient’s tumor burden via liquid biopsy. Liquid biopsies can assess ...a patient’s mutational landscape through time and provide information on treatment response and relapse. It has become increasingly common to have paired genomic data analysis between liquid and tissue biopsies from the same patient. However, the impact of clinical, temporal, and biologic factors on percentage of positive agreement (PPA) of variant detection between these biopsies is unclear. For our study, we leveraged two databases containing CGP data from paired liquid and tissue biopsy specimens tested by Foundation Medicine (FMI): the FMI database (>1700 paired samples) and the Flatiron Health-Foundation Medicine Clinico-Genomic Database (CGDB). The CGDB is a subset of the FMI database linked with the Flatiron Health nationwide de-identified EHR-derived database, which includes demographic, treatment, and clinical outcomes information (>500 paired samples). We report pan-solid tumor and per-indication prevalence and PPA for variants commonly assayed in both liquid and tissue tests. We found that PPA depended on the tumor DNA concentration in plasma and tissue biopsies, as well as the amount of time between tests. We also investigated the effect of treatments administered in the time between liquid and tissue tests on the detected variants and observed some well-described resistance alterations at a higher frequency in liquid samples, including a higher prevalence of ESR1 point mutations in breast cancer, more EGFR T790M alterations in lung cancer, and frequent KRAS Q61H alterations in colorectal cancer. Liquid biopsies can also contain DNA shed from multiple metastatic sites. We observed evidence of this in the form of polyclonal resistance alterations, which may also account for differences in PPA over time. Our findings indicate that while PPA is generally high between samples, it may be influenced by factors such as intervening therapies, resistance, therapy efficacy, and polyclonality of liquid samples.
Citation Format: Zoe June F. Assaf, Smruthy Sivakumar, Dexter X. Jin, Sophia L. Maund, Svetlana Lyalina, Guneet Walia, Ethan S. Sokol, Sally E. Trabucco. Pan-solid tumor comparison of variant detection in paired liquid and tissue biopsies abstract. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A17.
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Background: ctDNA can inform on prognosis, treatment response and survival. We evaluated ctDNA in serial plasma samples from patients enrolled in A.MARTIN (NCT01485861), a ...randomized phase II study of abiraterone with or without ipatasertib in patients with mCRPC. Methods: Blood was collected in cell-free DNA Streck tubes from 216 patients at 3 time points; baseline, C3D1 and end of treatment. Cell-free DNA (cfDNA) was extracted from plasma using a Circulating DNA Kit (Qiagen) on a QIASymphony machine (Qiagen). 25ng of extracted cfDNA was used in library preparation, constructed with a custom designed, 58 gene, QIAseq Targeted DNA panel (Qiagen) enriched for PI3K/AR pathway genes. Samples were sequenced to mean depth of 3394x on a NextSeq500 machine. Unless otherwise noted, all analyses combine patients across the 3 study arms, and reported p-values are unadjusted. Results: Baseline (BL) ctDNA positivity correlated with radiological progression-free survival (rPFS; HR: 1.8 95% CI 1.3-2.6, p < 0.01); this association with rPFS was maintained in a multivariate cox model with > 5 baseline clinical variables (HR: 1.6 95% CI 1.1-2.4; p = 0.011). Patients with a C3D1 reduction in ctDNA had superior rPFS compared to patients with a C3D1 increase in ctDNA (HR: 2 95% CI 1.3-3.2, p < 0.01). The rate of ctDNA clearance at C3D1 was higher in the Ipatasertib 400mg arm compared to placebo (56.3% versus 24.4%, p < 0.01). We find that changes in ctDNA associated with best confirmed overall response (p = 0.024); CR patients had the greatest reduction in ctDNA (mean of -23.4%), followed by PR (-16.3%), then SD (-4.1%), and lastly PD patients (-1.3%). Changes in ctDNA levels correlated with SLD changes (rs = 0.289, p = 0.05), and also PSA changes (rs = 0.33, p < 0.01). Changes in ctDNA were associated with rPFS in a multivariate cox analysis that included PSA change (p < 0.01), as well as in a separate multivariate analysis that included SLD change (p < 0.01). Lastly, we explored CNVs and observed emerging resistance mutations in progression samples, including alterations in TP53, AR, FOXA, PTEN, and PI3K/AKT pathway genes. Conclusions: ctDNA analyses may help (i) identify poorer prognosis disease at baseline, (ii) inform on treatment response (CR/PR/SD/PD) and radiological progression free survival (rPFS) in on-treatment (C3D1) samples, and (iii) can elucidate emerging resistance mechanisms at disease progression. Clinical trial information: NCT01485861 .
The protozoan parasite Giardia intestinalis (also known as Giardia lamblia) is a major waterborne pathogen. During its life cycle, Giardia alternates between the actively growing trophozoite, which ...has two diploid nuclei with low levels of allelic heterozygosity, and the infectious cyst, which has four nuclei and a tough outer wall. Although the formation of the cyst wall has been studied extensively, we still lack basic knowledge about many fundamental aspects of the cyst, including the sources of the four nuclei and their distribution during the transformation from cyst into trophozoite. In this study, we tracked the identities of the nuclei in the trophozoite and cyst using integrated nuclear markers and immunofluorescence staining. We demonstrate that the cyst is formed from a single trophozoite by a mitotic division without cytokinesis and not by the fusion of two trophozoites. During excystation, the cell completes cytokinesis to form two daughter trophozoites. The non-identical nuclear pairs derived from the parent trophozoite remain associated in the cyst and are distributed to daughter cells during excystation as pairs. Thus, nuclear sorting (such that each daughter cell receives a pair of identical nuclei) does not appear to be a mechanism by which Giardia reduces heterozygosity between its nuclei. Rather, we show that the cyst nuclei exchange chromosomal genetic material, perhaps as a way to reduce heterozygosity in the absence of meiosis and sex, which have not been described in Giardia. These results shed light on fundamental aspects of the Giardia life cycle and have implications for our understanding of the population genetics and cell biology of this binucleate parasite.
Abstract Introduction: Monitoring of ctDNA can be a minimally invasive complement to tumor imaging for assessing treatment effect. Technical limitations require methods to distinguish tumor signal ...from clonal hematopoiesis (CH), often including non-tumor sequencing. We developed a tumor-naïve panel (FoundationOne® Monitor) to quantify ctDNA tumor fraction (TF). TF analytical validation used peripheral blood mononuclear cell (PBMC) sequencing. We then leveraged plasma collected serially from patients with aNSCLC in the real-world (rw) Prospective Clinico-Genomic (PCG; NCT04180176) study to investigate the utility of TF for monitoring therapy (tx) response. Methods: TF was quantified using a combination of aneuploidy and variant allele frequencies of genomic alterations (GAs), while excluding CH mutations and aneuploidy using fragmentomic signal from cfDNA. In the PCG study, data from consenting patients were collected from electronic health records from 23 participating Flatiron Health Research Network sites. We analyzed plasma collected 6-15 weeks after start of tx in exploratory and validation cohorts per prespecified criteria. We defined molecular response (MR) as undetectable TF on tx regardless of baseline TF. Hazard ratios (HR) and 95% confidence Interval (CI) were calculated with univariate Cox proportional hazard regression. Results: For TF validation, 1135 samples separate from the PCG study with paired PBMC results were included. Overall, 24/4274 (0.56%) CH derived non-aneuploidy GAs detected in 1134 samples were falsely classified as somatic. Of these, 4 were detected among 317 samples with no tumor signal, resulting in a false positive TF value (specificity = 98.7%). CH derived aneuploidy was observed in 27 PBMCs and was appropriately filtered during TF estimation in all cases. Assessing the impact of CH aneuploidy filtering on sensitivity, we only identified 1/320 (0.31%) samples where non-CH derived aneuploidy was omitted. To assess clinical validity, 222 patients were analyzed from the PCG study. MR was assessed after a median of 10.8 weeks of tx (IQR 8.9-12.0). In a subset of 152 patients treated with physicians’ choice, MR was associated with favorable rw progression free survival (rwPFS: 9.4 v 2.8 months mo; HR = 0.30; 95% CI: 0.21-0.44) and rw overall survival (rwOS: 22.0 v 7.5 mo; HR = 0.33 0.22-0.49). We validated this finding on 70 patients receiving immunotherapy (50 with chemo). Again, MR was associated with favorable rwPFS (9.0 v 2.8 mo; HR = 0.28 0.16-0.51) and rwOS (20.2 v 9.4 mo; HR = 0.42 0.23-0.78). Conclusions: We describe a highly specific tumor naïve algorithmic filtration of non-tumor signal to enable high confidence ctDNA quantification and MR assessment. On tx MR is associated with favorable outcomes. These findings may enable personalized tx approaches tailored to a patient’s risk of progression and downstream cancer morbidity. Citation Format: Anne C. Chiang, Russell W. Madison, Yanmei Huang, Alexander Fine, Dexter X. Jin, Geoffrey R. Oxnard, Jason Hughes, Zoe June Assaf, Yi Cao, Vladan Antic, Ole Gjoerup, Amanda Young, David Fabrizio, Shaily Lakhanpal, Richard Zuniga, Katja Schulze, Lincoln W. Pasquina. Validation of a tumor-naïve circulating tumor DNA (ctDNA) response monitoring panel in advanced non-small cell lung cancer (aNSCLC) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 971.
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