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PURPOSE Anti–epidermal growth factor receptor (EGFR) antibodies are effective treatments for metastatic colorectal cancer. Improved understanding of acquired resistance mechanisms may facilitate circulating tumor DNA (ctDNA) monitoring, anti-EGFR rechallenge, and combinatorial strategies to delay resistance. METHODS Patients with treatment-refractory metastatic colorectal cancer (n = 169) enrolled on the CO.26 trial had pre–anti-EGFR tissue whole-exome sequencing (WES) compared with baseline and week 8 ctDNA assessments with the GuardantOMNI assay. Acquired alterations were compared between patients with prior anti-EGFR therapy (n = 66) and those without. Anti-EGFR therapy occurred a median of 111 days before ctDNA assessment. RESULTS ctDNA identified 12 genes with increased mutation frequency after anti-EGFR therapy, including EGFR ( P = .0007), KRAS ( P = .0017), LRP1B ( P = .0046), ZNF217 ( P = .0086), MAP2K1 ( P = .018), PIK3CG ( P = .018), BRAF ( P = .048), and NRAS ( P = .048). Acquired mutations appeared as multiple concurrent subclonal alterations, with most showing decay over time. Significant increases in copy-gain frequency were noted in 29 genes after anti-EGFR exposure, with notable alterations including EGFR ( P < .0001), SMO ( P < .0001), BRAF ( P < .0001), MET ( P = .0002), FLT3 ( P = .0002), NOTCH4 ( P = .0006), ERBB2 ( P = .004), and FGFR1 ( P = .006). Copy gains appeared stable without decay 8 weeks later. There were 13 gene fusions noted among 11 patients, all but one of which was associated with prior anti-EGFR therapy. Polyclonal resistance was common with acquisition of ≥ 10 resistance related alterations noted in 21% of patients with previous anti-EGFR therapy compared with 5% in those without ( P = .010). Although tumor mutation burden (TMB) did not differ pretreatment ( P = .63), anti-EGFR exposure increased TMB ( P = .028), whereas lack of anti-EGFR exposure resulted in declining TMB ( P = .014). CONCLUSION Paired tissue and ctDNA sequencing identified multiple novel mutations, copy gains, and fusions associated with anti-EGFR therapy that frequently co-occur as subclonal alterations in the same patient.

PURPOSEAlthough the majority of patients with metastatic non–small-cell lung cancer (mNSCLC) lacking a detectable targetable mutation will receive pembrolizumab-based therapy in the frontline setting, predicting which patients will experience a durable clinical benefit (DCB) remains challenging.MATERIALS AND METHODSPatients with mNSCLC receiving pembrolizumab monotherapy or in combination with chemotherapy underwent a 74-gene next-generation sequencing panel on blood samples obtained at baseline and at 9 weeks. The change in circulating tumor DNA levels on-therapy (molecular response) was quantified using a ratio calculation with response defined by a > 50% decrease in mean variant allele fraction. Patient response was assessed using RECIST 1.1; DCB was defined as complete or partial response or stable disease that lasted > 6 months. Progression-free survival and overall survival were recorded.RESULTSAmong 67 patients, 51 (76.1%) had > 1 variant detected at a variant allele fraction > 0.3% and thus were eligible for calculation of molecular response from paired baseline and 9-week samples. Molecular response values were significantly lower in patients with an objective radiologic response (log mean 1.25% v 27.7%, P < .001). Patients achieving a DCB had significantly lower molecular response values compared to patients with no durable benefit (log mean 3.5% v 49.4%, P < .001). Molecular responders had significantly longer progression-free survival (hazard ratio, 0.25; 95% CI, 0.13 to 0.50) and overall survival (hazard ratio, 0.27; 95% CI, 0.12 to 0.64) compared with molecular nonresponders.CONCLUSIONMolecular response assessment using circulating tumor DNA may serve as a noninvasive, on-therapy predictor of response to pembrolizumab-based therapy in addition to standard of care imaging in mNSCLC. This strategy requires validation in independent prospective studies.

Purpose: Tumor mutational burden (TMB) has been shown to be predictive of survival benefit in patients with non–small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors. Measuring TMB in the blood (bTMB) using circulating cell-free tumor DNA (ctDNA) offers practical advantages compared with TMB measurement in tissue (tTMB); however, there is a need for validated assays and identification of optimal cutoffs. We describe the analytic validation of a new bTMB algorithm and its clinical utility using data from the phase III MYSTIC trial. Patients and Methods: The dataset used for the clinical validation was from MYSTIC, which evaluated first-line durvalumab (anti–PD-L1 antibody) ± tremelimumab (anticytotoxic T-lymphocyte-associated antigen-4 antibody) or chemotherapy for metastatic NSCLC. bTMB and tTMB were evaluated using the GuardantOMNI and FoundationOne CDx assays, respectively. A Cox proportional hazards model and minimal P value cross-validation approach were used to identify the optimal bTMB cutoff. Results: In MYSTIC, somatic mutations could be detected in ctDNA extracted from plasma samples in a majority of patients, allowing subsequent calculation of bTMB. The success rate for obtaining valid TMB scores was higher for bTMB (809/1,001; 81%) than for tTMB (460/735; 63%). Minimal P value cross-validation analysis confirmed the selection of bTMB ≥20 mutations per megabase (mut/Mb) as the optimal cutoff for clinical benefit with durvalumab + tremelimumab. Conclusions: Our study demonstrates the feasibility, accuracy, and reproducibility of the GuardantOMNI ctDNA platform for quantifying bTMB from plasma samples. Using the new bTMB algorithm and an optimal bTMB cutoff of ≥20 mut/Mb, high bTMB was predictive of clinical benefit with durvalumab + tremelimumab versus chemotherapy.

Background: Circulating tumor DNA (ctDNA) level and the change in level at a subsequent time point (e.g. on-treatment change from baseline or postoperative changes through time) are promising tools for predicting patient prognosis and response to therapy. Existing methods use somatic variant allele frequencies to quantify circulating tumor fractions (cTF). Their performance can be limited by the number of detectable somatic alterations and the associated limit of detection (LoD), as well as interference from copy number variation and non-tumor alterations, such as clonal hematopoiesis. Here, we describe the LoD, precision and limit of quantitation (LoQ) of cTF level and change using GuardantINFINITY, a next generation sequencing panel covering over 800 genes with genome-wide methylation detection. Method: The cTF of a single sample is estimated from methylation signals across targeted regions of the GuardantINFINITY methylation panel, calibrated using internal training data. cTF change compares two or more samples from the same patient to identify patient-specific methylated regions, and compare the methylation signals of the paired regions. LoQ of cTF level and change were assessed in experimental titrations of advanced colorectal, breast, and lung cancer patient samples and cell line samples into cancer-free backgrounds at different target levels between 0.1%-0.5% cTF. LoD is defined as the lowest cTF level where >95% replicates were detected to have tumor-derived methylation signals. LoQ of cTF level or change is defined as the lowest cTF where the coefficient of variation (CV) across replicates is less than 30%. Accuracy of methylation based cTF compared to cTFs calculated from maximum VAF of somatic mutations was assessed on 1,400 clinical samples of colorectal and lung cancer patients (N=189, 372, 252 and 463 for stage I to IV). Results: Experimental titrations of cancer samples demonstrated a single-sample LoD of 0.05% cTF (lowest dilution level) and quantitative precision down to a LoQ of below 0.1%, compared to the LoQ of 0.3% estimated by somatic mutations. In paired clinical titration samples, the LoQ of methylation ctDNA level change was also below 0.1%, compared to the LoQ of ctDNA level change estimated by somatic mutations at 0.3-0.5%. In the 1,400 clinical samples, 64% had at least one somatic mutation detected, 90% had ct-DNA detected with methylation and 96% of these ct-DNA detected samples had cTF above the defined methylation LoQ. Among patients with both methylation and genomic signals identified, the methylation method quantified a similar cTF to those that were calculated using maximum somatic driver mutations (Pearson r=0.83). Conclusion: Methylome sequencing using GuardantINFINITY enables accurate and precise quantification of ctDNA level and change with a liquid-only approach, offering longitudinal ctDNA monitoring for more patients than previous methods. Citation Format: Sai Chen, Katie Quinn, Che-Yu Lee, Jun Zhao, Kyle Chang, Tingting Jiang, Shile Zhang, Carin Espenschied, Sara Wienke, Thereasa Rich, Indira Wu, Yvonne Kim, Xianxian Liu, Nageswara Alla, Dustin Ma, Giao Tran, Han-Yu Chuang. A method for quantifying circulating tumor DNA level and molecular response using methylome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3123.

Background: Liquid biopsy has been established as a powerful, non-invasive means to profile tumors in and identify clinically relevant variants. However, the presence of clonal hematopoiesis (CH) variants, or biological noise, due to aging or therapy has potential to confound biomarker interpretation. Currently, comprehensive methods to filter out non-tumor variants require genotyping the white blood cell (WBC) fraction of the paired plasma sample, which is a costly, complicated workflow. A plasma-only, bioinformatics solution to identify non-tumor variants is needed for accurate biomarker assessments in the cell-free DNA (cfDNA). Method: An ensemble model was trained on a database of >250,000 plasma samples comprising healthy donor, early and late-stage cancer patients sequenced on the Guardant360TM , GuardantREVEALTM, and GuardantOMNITM liquid biopsy panels as well as public tissue datasets. The model was optimized with 5 fold cross-validation and hyperparameter tuning to produce a non-tumor and tumor variant classifier. To validate these calls, 116 paired plasma and WBC advanced cancer samples were selected for high prevalence of putative CH variants and genotyped using an in-house bioinformatics pipeline. In the validation cohort, cfDNA variants were determined to be of non-tumor or CH origin if there was molecule support in the WBC; cfDNA variants above 0.6% (limit of detection in the gDNA) with no support in the WBC were determined to be from the tumor. Results: The validation cohort consisted of 2150 somatic SNV and Indels, 956 of which were confirmed in the WBC and 1194 confirmed as plasma-only. Half of confirmed CH variants (48%, 458/956) occurred in known CH genes, while the other half occurred in genes such as TP53, ATM, NOTCH4, FAT1, SRSF2. No clinically actionable variants were confirmed in the WBC. Non-tumor or CH predictions were made for 624 somatic variants: 515/624 correctly identified CH, for a positive predictive value (PPV) of 83%. Of all CH variants confirmed in the WBC, 54% (553/956) had a CH or non-CH prediction; CH predictions had 91% (515/553) positive percent agreement (PPA) with the WBC. Remaining variants with no CH prediction (403/956) were low or no prevalence across datasets and occurred predominantly in LRP1B, TET2, TP53, KMT2D. Nearly half (67%, n=109) of CH predictions not in WBC occurred in a CH gene. For non-CH gene variants, 16% of false positive predictions occurred in 6 variants across 4 genes (ACVR2A, RNF43, B2M, FLT3). Conclusion: We present a plasma-only method that has high PPA and PPV with WBC genotyping for classifying non-tumor, CH variants in the cfDNA. Further investigation is underway to improve the sensitivity of annotating rare CH variants. Accurate CH identification is critical for treatment selection across targeted therapies, particularly for loss of function variants in DNA repair genes that may confer sensitivity to PARPi or ATRi therapies. Citation Format: Jennifer Yen, Yu Fu, Jeff Werbin, Katie Quinn, Robert Foley, Minh Trahn, Carin Espenschied, Scott Higdon, Brett Kennedy, Han-Yu Chuang. Validation of a bioinformatic model for classifying non-tumor variants in a cell-free DNA liquid biopsy assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3135.

Introduction: Genomic tests are routinely used by clinicians to guide treatment decisions in early-stage breast cancer (EBC). The 70-gene MammaPrint assay (MP) assesses the risk of distant recurrence in untreated patients with EBC and categorizes the tumors as High Risk (HR, MP index: -1 to ≤0) or Low Risk (LR, >0 to +1). The LR category is further divided into Low but non-UltraLow (LNUL; >0 to ≤0.355) and UltraLow Risk (UL; >0.355 to 1). Here, we report the risk of distant recurrence by MP and 10-year outcomes in patients with EBC diagnosed at Magee Women’s Hospital of the University of Pittsburgh Medical Center. Methods: In this retrospective analysis, 259 women diagnosed with EBC between 2005 and 2008, who received a MP result, were included. Patient clinical and tumor characteristics were collected. The median FU was 13.1 year among patients with clinical data. Treatment received, 10-year Distant Metastasis Free Interval (DMFI) and 10-year Breast Cancer Specific Survival (BCSS) are reported according to the MP groups. Differences in DMFI and BCSS between MP risk groups were assessed by log-rank. Patients were treated at the physician’s discretion. Treatment was started prior to obtaining MammaPrint results. Results: Among the 259 patients, 69% were post-menopausal women (mean [range] age: 58 [31-81] years) and diagnosed with hormone receptor-positive HER2-negative tumors (90%), grade 1 or 2 (64%), and without lymph node invasion (93%). In this cohort, 69% (n = 159) had a MP LR result and 31% (n = 100) had a MP HR result. Overall, 14% (n = 35) of patients had a MP UL risk of recurrence of whom 74% (n = 26/35) were post-menopausal women. Substantially more patients received chemotherapy in the HR group (57%, n = 57) compared with the LR group (15%, n = 24) (table). Considering that treatment was initiated before MammaPrint results were known, MP results might have allowed chemotherapy de-escalation in in 15% (n = 24) of patients with a MP LR. Similarly, in the 39% (n = 39) of women with a MP HR treated with endocrine therapy only, knowledge of MP results could have provided important information to add chemotherapy to the treatment plan.. In the MP UL group, 74 % (n = 26) of patients were treated with endocrine therapy only compared with those who received chemotherapy (9%, n = 3) and no adjuvant treatment (9%, n = 3). The 10-year DMFI and 10-year BCSS were higher in the LR compared with the HR group (table). When further stratifying the MP LR group in LNUL and UL, the 10y DMFI was 0.97 (95% CI; 0.94 – 1.00) and 1.00 for the MP LNUL and UL groups, respectively. Within the first 10 years, 8 of the 10 distant recurrences observed were in the MP HR group, and 2 were in the MP LNUL group. Among the 18 recorded deaths, 5 were breast cancer-related, 4 in the MP HR and 1 in the MP LR (LNUL) groups. Discussion: In this single-institution retrospective analysis, all patients showed excellent BCSS and DMFI outcomes confirming the ability of MP to correctly predict the good prognosis (LR) and poor prognosis (HR) in patients with EBC. In this analysis, as observed in other cohorts, women with a MP UL risk result had an excellent prognosis at 10 years while being treated mostly with endocrine therapy only. Taken together, with the low endocrine therapy adherence reported in the literature, these data suggest that patients with a MP UL result may be candidates for further treatment de-escalation to optimize the risk/benefit ratio of endocrine therapy in future studies. Table 1. Clinical outcomes and treatment received in patients stratified by MammaPrint results * Patients were treated at the physician’s discretion a p = 0.011, MP LR vs MP HR. b p = 0.032, MP UL vs LNUL vs HR. c p = 0.061 MP LR vs MP HR. d p = 0.170, MP UL vs LNUL vs HR. Citation Format: Azadeh Nasrazadani, Juan L. Gomez Marti, Margaret Q. Rosenzweig, Meghan McGuire, Katie Quinn, Josien Haan, Alexandre Houzelle, Rohit Bhargava, William Audeh, Adam M. Brufsky. Utility of the 70-gene signature and 10 year follow up in patients with early-stage breast cancer in a single institution study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-20.

Background: Precision profile simulations (PPS) can be used to assess variability of biomarker profiles and provide valuable insight into assay performance, especially when reliable precision estimates can not be obtained empirically due to scarcity of representative samples or insufficient materials per sample. A PPS was conducted for the GuardantOMNI assay to characterize the expected variability in blood tumor mutational burden (bTMB) score across a representative range of expected bTMB scores in clinical samples. The simulations were aligned to, but not completely prescribed by, the PPS guidance provided in Guidance for Industry and and Food and Drug Administration Staff Class II Special Controls Guidance Document: Ovarian Adnexal Mass Assessment Score Test System. A sample’s bTMB score is a real valued quantity (e.g., bTMB = 21.04 mut/Mb) that is derived by multiplying the number of qualified mutations observed within a targeted panel by a scaling factor. Variability in observed bTMB scores for a given blood sample is governed primarily by sample coverage, tumor shedding level, and the assay somatic variant detection probabilities (a function of underlying variant allele frequencies, VAFs). Methods: The relationship between site-specific total molecule counts and coverage was modeled utilizing a composite dataset consisting of both clinical and contrived samples. Sample coverage was modeled using variance component estimates from Precision Study data (18 cancer samples each with 6 to 18 replicates). The reference, single-strand mutant, and double-strand mutant molecule counts for a somatic variant site detected in at least one sample replicate were modeled utilizing a bias corrected Dirichlet Multinomial model. The variants with the simulated VAF and coverage levels were processed with the GuardantOMNI germline/somatic classifier to account for the uncertainty in germline/somatic classification at lower coverage values. Results: Precision profiles consisting of simulation derived %CV estimates for 18 clinical samples with a representative set of mean bTMB scores were generated. The PPS bTMB score distributions were consistent with the bTMB scores observed in the Precision Study, supported by visualization and confidence intervals at level 0.05 margins of equivalence for the empirical mean bTMB and standard deviation estimates. The sample specific %CV estimates were observed, in most instances, to decrease with increasing input levels for matched targeted LoD (Limit of Detection) simulation results. Precision profile %CV estimates were observed to be inversely related to mean bTMB scores. Conclusions: The results provide proof of principle that estimation of GuardantOMNI bTMB score precision via an intuitive and interpretable simulation model is viable. The simulation results were consistent with empirical data and general expectations regarding the precision of the bTMB scores. Citation Format: Kevin Doubleday, Daniel Gaile, Ravi Vijaya-Satya, Xianxian Liu, Kevin D'Auria, Soni Shukla, Han-Yu Chuang, Katie Quinn, Darya Chudova. Precision profile simulation study for a next generation sequencing bTMB assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5015.

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BackgroundTumor mutational burden (TMB), defined as the number of somatic mutations per megabase of interrogated genomic sequence, demonstrates predictive biomarker potential for the identification of patients with cancer most likely to respond to immune checkpoint inhibitors. TMB is optimally calculated by whole exome sequencing (WES), but next-generation sequencing targeted panels provide TMB estimates in a time-effective and cost-effective manner. However, differences in panel size and gene coverage, in addition to the underlying bioinformatics pipelines, are known drivers of variability in TMB estimates across laboratories. By directly comparing panel-based TMB estimates from participating laboratories, this study aims to characterize the theoretical variability of panel-based TMB estimates, and provides guidelines on TMB reporting, analytic validation requirements and reference standard alignment in order to maintain consistency of TMB estimation across platforms.MethodsEleven laboratories used WES data from The Cancer Genome Atlas Multi-Center Mutation calling in Multiple Cancers (MC3) samples and calculated TMB from the subset of the exome restricted to the genes covered by their targeted panel using their own bioinformatics pipeline (panel TMB). A reference TMB value was calculated from the entire exome using a uniform bioinformatics pipeline all members agreed on (WES TMB). Linear regression analyses were performed to investigate the relationship between WES and panel TMB for all 32 cancer types combined and separately. Variability in panel TMB values at various WES TMB values was also quantified using 95% prediction limits.ResultsStudy results demonstrated that variability within and between panel TMB values increases as the WES TMB values increase. For each panel, prediction limits based on linear regression analyses that modeled panel TMB as a function of WES TMB were calculated and found to approximately capture the intended 95% of observed panel TMB values. Certain cancer types, such as uterine, bladder and colon cancers exhibited greater variability in panel TMB values, compared with lung and head and neck cancers.ConclusionsIncreasing uptake of TMB as a predictive biomarker in the clinic creates an urgent need to bring stakeholders together to agree on the harmonization of key aspects of panel-based TMB estimation, such as the standardization of TMB reporting, standardization of analytical validation studies and the alignment of panel-based TMB values with a reference standard. These harmonization efforts should improve consistency and reliability of panel TMB estimates and aid in clinical decision-making.

When People Die: Stories from Young People is a comic that tells numerous stories about death and resilience from a group of young people. The comic helps readers gain different and better perspectives on grief and what grieving means for young people. These stories and scenarios have been written by a group of young people selected from Children’s Hospices Across Scotland (Robin House), HMYOI Polmont, and Richmond’s Hope, and put together by the team at the Dundee Comics Creative Space. This comic will help people such as school teachers, guidance counsellors and anyone who reads it to learn more about how it feels to be in the position of a grieving young person, and how to act in situations that may come up with a grieving child.

Purpose: The role of plasma-based tumor mutation burden (pTMB) in predicting response to pembrolizumab-based first-line standard-of-care therapy for metastatic non–small cell lung cancer (mNSCLC) has not been explored. Experimental Design: A 500-gene next-generation sequencing panel was used to assess pTMB. Sixty-six patients with newly diagnosed mNSCLC starting first-line pembrolizumab-based therapy, either alone or in combination with chemotherapy, were enrolled (Clinicaltrial.gov identifier: NCT03047616). Response was assessed using RECIST 1.1. Associations were made for patient characteristics, 6-month durable clinical benefit (DCB), progression-free survival (PFS), and overall survival (OS). Results: Of 66 patients, 52 (78.8%) were pTMB-evaluable. Median pTMB was 16.8 mutations per megabase (mut/Mb; range, 1.9–52.5) and was significantly higher for patients achieving DCB compared with no durable benefit (21.3 mut/Mb vs. 12.4 mut/Mb, P = 0.003). For patients with pTMB ≥ 16 mut/Mb, median PFS was 14.1 versus 4.7 months for patients with pTMB < 16 mut/Mb [HR, 0.30 (0.16–0.60); P < 0.001]. Median OS for patients with pTMB ≥ 16 was not reached versus 8.8 months for patients with pTMB < 16 mut/Mb [HR, 0.48 (0.22–1.03); P = 0.061]. Mutations in ERBB2 exon 20, STK11, KEAP1, or PTEN were more common in patients with no DCB. A combination of pTMB ≥ 16 and absence of negative predictor mutations was associated with PFS [HR, 0.24 (0.11–0.49); P < 0.001] and OS [HR, 0.31 (0.13–0.74); P = 0.009]. Conclusions: pTMB ≥ 16 mut/Mb is associated with improved PFS after first-line standard-of-care pembrolizumab-based therapy in mNSCLC. STK11/KEAP1/PTEN and ERBB2 mutations may help identify pTMB-high patients unlikely to respond. These results should be validated in larger prospective studies.

Background/Study Context: Older adults show a greater response to feedback whilst learning than younger adults. To date this has only been shown for receiving veridical feedback, but there is evidence that suggests that receiving false positive feedback may further enhance learning. We tested the hypothesis that receiving false positive feedback, being told you are preforming better than expected, would be more advantageous for older than younger adults when learning an inhibitory-action task. Methods: 42 younger and 34 older adults trained to improve their inhibition and response times on the Simon task. They completed 18 training blocks and a retention test two weeks after training. Participants received either false positive feedback or veridical feedback on their performance at the end of each training session and the start of the next session. Those in the false positive feedback group were told they were performing faster than expected. Results: Both older and younger adults improved their inhibition and response times but receiving false positive feedback did not significantly change their rate of learning on these outcomes. However, false positive feedback did impact on accuracy levels with those receiving this type of feedback making fewer errors. Older adults were slower but more accurate than younger adults, but contrary to our hypothesis they did not benefit more from false positive feedback than younger adults. Conclusion: This first direct comparison of the effects of false positive feedback on older and younger adults showed that the positive impact of false positive feedback does not decline with age. We also demonstrated that feedback given about one aspect of a skill (in this case speed) may in fact influence another aspect of the skill (in this case accuracy). This suggests that false positive feedback could be used as a motivational tool to enhance cognitive-motor learning in older adults, but care needs to be taken when using this, as the feedback may not affect the element of the skill at which it is targeted.

Background: Molecular response (MR) estimated as a change in circulating tumor (ctDNA) load between an early on treatment sample (usually 2-9 weeks post treatment start) and pre-treatment baseline has been shown to predict patient response and outcomes across solid tumors and therapy types in many retrospective studies. There is no consensus, however, regarding the best method for assessing molecular response. Therefore, we aimed to assess several molecular response calculations and determine the optimal method for predicting outcomes in individual advanced cancer patients. Method: Aggregate results of > 4,000 patient sample pairs (3-10 weeks apart), > 1000 patient sample technical replicates, > 100 contrived sample dilutions, and in silico simulations were analyzed with Guardant360TM or GuardantOMNITM. Baseline and on-treatment paired patient samples were collected from advanced cancer patients with over 12 tumor types, including lung, colon, and breast. MR calculations included variant allele fractions (VAFs) of somatic SNVs, indels and fusions. Methods including Ratio treatment/baseline (R) of Maximum VAF (maxVAF), Ratio of Mean VAF (mVAF), and Mean of VAF Ratios were compared, with consideration of VAF precision. Analytical accuracy, reproducibility and limit of detection (LoD) were assessed. Results: Comparison of methods for calculating net change in ctDNA load on > 1500 sample pairs showed high correlation (ρ ranged from 0.93 to 0.98) and categorical agreement split by the median (93%). Therefore selecting an optimal method based on outcome prediction would require prohibitively large patient cohorts. Analytical evaluation and in silico simulations can predict the behavior of each method. Simulations of changes in tumor fraction of real pre-treatment samples found that RmVAF or RmaxVAF are more accurate than mVAFR, which can be skewed by low VAF ratios. Almost 25% of sample pairs have a tumor driver or resistance mutation that is not the maxVAF, suggesting tumor dynamics are better captured by mVAF than maxVAF. Newly-detected on-treatment variants can be an important signal of rising ctDNA levels, impacting MR in approximately 2% of sample pairs. Importantly, MR accuracy for all methods decreases as maxVAF approaches or falls below the variant LoD, due to both stochastic detection and higher CV of variants at low VAF. Thus the assay variant LoD is a key determinant of the fraction of patients who can receive MR evaluation. Technical replicates identified the variant criteria at which a 50% change in tumor fraction differs significantly from technical variation, and could define analytical reporting limits. Conclusions: Comparison of MR methods in a large set of patient samples and simulations supports RmVAF with inclusion of newly-detected mutations. Clinical validation of these methods will support patient-specific MR prediction of outcomes. Citation Format: Allysia J. Mak, Katie J. Quinn, Carin Espenschied, Kyle Chang, Han-Yu Chuang, Elena Helman, Darya Chudova, Justin Odegaard, Becky Nagy, AmirAli Talasaz. Comparison of molecular response calculations for prediction of patient outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 401.

5038 Background: Accurate analysis of the genomic alteration landscape within tissue- and plasma-derived tumor DNA using next-generation sequencing (NGS) may provide insights into specific patient populations that benefit from different therapies. The interchangeable use of tissue- and plasma-based assessments may benefit patients when tissue availability is limited, a common occurrence in individuals with mCRPC. To understand the potential sources of technical and biological variability in this setting, we performed comprehensive comparative analyses across 3 NGS platforms, using samples from patients enrolled in CheckMate 9KD, a phase 2 study of nivolumab combined with docetaxel, rucaparib, or enzalutamide for patients with confirmed mCRPC (NCT03338790). Methods: We performed retrospective integrated analyses of sequence and structural alterations identified through comprehensive genomic profiling (CGP) of DNA obtained from formalin-fixed, paraffin-embedded tissue specimens and cell-free DNA obtained from plasma. Tissue-based analysis was performed using the FoundationOne assay (F1, 395 genes), while the FoundationACT (FACT, 70 genes) and GuardantOMNI (OMNI, 500 genes) assays were used for plasma-based analysis. Analysis was performed on samples from 103 patients for which datasets from all 3 assays were available. Inter-platform analysis considered variants with ≥ 0.50% variant allele fraction and common to the shared pairwise targeted regions, while excluding synonymous variants. Results: Through broad profiling of DNA obtained from tissue and plasma, we uncovered previously identified recurrent alteration of AR, TP53, PTEN, and TMPRSS2 fusion with ETS genes. Additionally, we found that 42% (F1), 45% (FACT), and 34% (OMNI) of patients harbored a combination of germline and somatic mutations in homologous recombination repair pathway genes. Across all samples, median tumor mutational burden was 3.5 mutations per megabase (mut/Mb) by F1 and 8.6 mut/Mb by OMNI. Inter-platform variant analyses demonstrated concordance of 52% for F1 vs FACT, 40% for F1 vs OMNI, and 75% for FACT vs OMNI. Conclusions: Overall, these data demonstrate the value of integrated tissue and liquid biopsy profiling in mCRPC. Both technical and biological sources of variation, including panel size, mutation detection algorithms, variant annotation and reporting, analytical performance, circulating tumor DNA levels, and tumor heterogeneity, may be captured differently by tissue- and plasma-based techniques, accounting for the discordance in reported results. Clinical trial information: NCT03338790.

61 Background: Pembrolizumab was recently granted tissue agnostic FDA accelerated approval for metastatic cancers with TMB≥10 mut/Mb. However, limited data supports immunotherapy in microsatellite stable (MSS) mCRC with TMB≥10 mut/Mb. We assessed tissue TMB and contrasted it to plasma derived TMB in the CO.26 trial. Methods: CO.26 was a phase 2 trial (2-sided ⍺ = 0.1 and 80% power) that randomized 180 patients (pts) 2:1 to D+T or BSC in refractory mCRC. Pre-treatment plasma was sequenced with the GuardantOMNI assay and archival tissue underwent exome sequencing with TMB assessed per the TMB harmonization project. MSI-H cases were excluded. For plasma TMB, we used a previously published cut point (≥28). Results: Overall survival (OS) but not progression free survival (PFS) was improved with D+T in the entire population. Of 180 pts, 163 were evaluable for plasma and 110 for tissue TMB. Median time between archival tissue and plasma collection was 3.1 yrs (IQR 1.9-5.1). Median tissue TMB was 6.6 muts/Mb (IQR 4.1-12.0), while median plasma TMB was 16.3 muts/Mb (IQR 9.4-25.9). Tissue and plasma TMB (r = -0.039, P = 0.69) were not correlated. Tissue TMB≥10 was not prognostic in the BSC arm (HR 1.01, 90%CI 0.52-1.92, P = 0.99) and OS was not improved in pts with tissue TMB≥10 (32/110 pts) following D+T vs BSC. A test of interaction suggested this threshold was not predictive (P = 0.85). Using a minimum P-value approach, no threshold supported high tissue TMB as predictive in MSS mCRC. In fact, the optimal cut point suggested low tissue TMB ( < 4.1 muts/Mb) had the greatest benefit from D+T (P-interaction = 0.048) and pts with TMB ≥4.1 mut/Mb (HR 0.50, 90%CI 0.26-0.96, P = 0.083) trended to better OS in the BSC arm. In contrast, 35/163 pts (21%) were identified in a high plasma TMB group associated with worse OS (HR 2.56, 90%CI 1.45-4.54, P = 0.007) in the BSC arm but improved OS following D+T compared to BSC with P-interaction = 0.082. Only 1 response was noted following D+T in a pt with tissue TMB = 16 mut/Mb and plasma TMB = 13 mut/Mb. Conclusions: Archival tissue TMB≥10 mut/Mb does not appear predictive of D+T benefit in MSS mCRC. Plasma derived TMB may better reflect evolutionary changes following intervening therapy than archival tissue. Clinical trial information: NCT02870920. [Table: see text]

Background: Clonal hematopoiesis (CH) is the acquisition of mutations in hematopoietic progenitor cells that can lead to clonal expansion. Recent studies suggest that CH-derived mutations can confound interpretation of cell-free DNA (cfDNA) sequencing results. To better understand the contribution of CH to cfDNA analysis in the metastatic cancer setting, we characterized CH-associated alterations observed in the cfDNA of late-stage cancer patients. Methods: We analyzed somatic variants from cfDNA profiles of over 62,000 patients in four late-stage cohorts: lung (>35,000), gastro-intestinal (>14,000), urogenital (>4,700), breast (>8,600). Matched white blood cell (WBC) and cfDNA was obtained for a subset of patient samples. Plasma cfDNA was processed using a 73-gene (150Kb, Guardant360TM) or 500-gene (2Mb, GuardantOMNITM) panel and sequenced to average depth of ~5000 molecules, with a 95% limit of detection (LOD) of 0.3-0.4% and 0.15-0.6% variant allele fraction (VAF) for SNVs, and 0.2%-0.7% and 0.4-0.8% for indels (for G360 and OMNI, respectively). Results: In samples processed on the 500-gene panel, DNMT3A, TET2, PPM1D, ASXL1 and SF3B1 were the most frequently mutated CH-associated genes (75% of samples). While the majority (73%) of these mutations were at low variant allele fractions (VAF) of 60,000 samples, JAK2 V617F, a known CH mutation, was observed in 927 samples with VAFs between 0.29% and 98%. Across the combined cohort, we found that in samples with a known CH variant (n=7,717), half of samples had a max CH VAF less than 50% of max tumor VAF. Interestingly, in >25% of samples, the max CH VAF was higher than the max tumor VAF. Initial comparisons of matched plasma and WBC DNA show that 100% (16/16) of clinically relevant and 92% (84/91) of variants of unknown significance (VUSs) across the 73-gene panel were found exclusively in plasma DNA and not in WBC DNA testing. Conclusions: We characterize the distribution of CH mutations across a large number of late-stage plasma samples and show that within highly pre-treated metastatic patients, the VAFs of CH mutations often differ from the tumor and can surpass the level of tumor shedding in circulation. Further investigation of these variants will enable improved understanding of CH in metastatic disease and its differentiation from the circulating tumor DNA. Citation Format: Jennifer Yen, Katie Quinn, Elena Helman, Andrey Chursov, Tracy Nance, Ariel Jaimovich, Kimberly Banks, Aleksandra Franovic, Kristin Gleitsman, John Latham, Arielle Yablonovitch, Marcin Sikora, Stephen Fairclough, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. Analysis of clonal hematopoiesis-associated mutations in the cell-free DNA of advanced cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2509.

Background: Tumor mutational burden (TMB) has emerged as a predictive biomarker of response to immune checkpoint inhibitor (ICI) therapy. Current panel-based TMB algorithms aggregate signal from certain types of somatic variants (e.g. non-synonymous coding SNVs); however, delineating the contributions of these and other types of mutations may refine TMB calculation from gene panels. Moreover, early studies suggest other possible genomic correlates of patient outcome to ICI which may be complementary to TMB. Here, we explore the landscape of mutations comprising TMB and other genomic features correlating with TMB on a subset of several thousand late-stage plasma samples run on GuardantOMNITM (OMNI), a highly sensitive 500-gene cfDNA sequencing platform. Methods: We developed a cfDNA-based TMB algorithm which is robust to variable tumor shedding levels and presence of clonal hematopoiesis. We assessed cfDNA-based TMB in over 1,000 plasma samples across six tumor types, including lung and prostate. We examine the contribution of nonsynonymous, synonymous, intronic SNVs, and indels to TMB score. We investigate correlations between TMB and additional genomic features, including chromosomal instability, loss of HLA-bearing chromosome 6p, microsatellite instability (MSI), and common oncogenic and resistance mutations. Results: We found that the distribution of WES-calibrated TMB scores across this cohort of samples is consistent with TCGA, with median 10 mutations/Mb and upper-tertile of 14 mutations/Mb across tumor types. The number of non-synonymous coding SNVs per sample correlated highly with synonymous coding SNV and intronic SNV counts (Pearson’s r > 0.7 for each). Including this additional signal in TMB calculation improves clinical sensitivity by up to 5%. In MSS samples, indels were highly correlated with SNVs, indicating that both likely arise from a similar underlying mechanism. We found no clear correlation between high TMB and chromosomal instability, with high TMB samples exemplifying a range of tumor ploidies. TMB association with oncogenic drivers is consistent with existing literature, with lower median TMB in EGFR-driven lung tumors (p < 0.01), but little to no correlation between TMB and KRAS or PIK3CA driver status, or STK11 loss of function (p > 0.05), suggesting these latter events could be independent clinical biomarkers to TMB. Conclusions: Panel-based TMB scores can leverage synonymous and non-coding mutations to strengthen the signal of exome-wide mutation load. As more patient outcome data becomes available, TMB algorithms and orthogonal biomarkers of tumor genome immunogenicity will evolve further for improved guidance of patient response to immunotherapy. Sequencing panels with high sensitivity for TMB, via large panel space, and the ability to detect copy-number variations and MSI-status, will be important for biomarker development and clinical applications. Citation Format: Katie Quinn, Elena Helman, Tracy Nance, Jennifer Yen, John Latham, Kristin Gleitsman, Ravi Vijaya-Satya, Carlo Artieri, Alex Artyomenko, Marcin Sikora, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. Landscape and genomic correlates of ctDNA-based tumor mutational burden across six solid tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3404.

3552 Background: MSI-H cancers are responsive to immune checkpoint blockade (ICB), but nearly half of all patients experience primary or early treatment resistance. Activation of the WNT/B-Catenin pathway can lead to immune exclusion and may drive resistance to ICB. Methods: 12 patients had stage III (N = 1) or IV (N = 11) MSI-H GI tract (small bowl, colon, or rectal) cancers. Blood samples were obtained after (N = 5) or during (N = 5) ICB. 2 patients did not receive ICB. Blood samples from 8 patients with microsatellite stable (MSS) metastatic colorectal cancer were included as controls. The Guardant Health (Redwood City, CA) Omni 2.0 mb panel was used to analyze cfDNA. We analyzed MSI-H status, TMB, and mutations within the WNT/B-Catenin pathway, including APC, RNF43 and CTNNB1. Results: Of 12 patients with MSI-H GI cancers, 1 sample failed enrichment due to hemolysis. MSI-H was not detected in 2 patients with a history of MSI-H in tissue; however these patients had a complete response to ICB at the time of blood collection. The Omni panel identified MSI-H in the remaining 9 patients with MSI-H disease in tissue. Among 8 control patients with MSS disease in tissue, MSI-H was not detected. Median TMB (mutations/Mb) was greater for MSI-H specimens (109; range 30-807) than for MSS specimens (13; range 6-24). All 8 patients with MSS GI cancers were identified to have APC mutations, and none were found to have CTNNB1 or RNF43 mutations. Of 9 evaluable MSI-H GI cancers, 2 had APC mutations alone. The remaining 7 carried RNF43 mutations (G659fs). All patients with RNF43 mutations were found to have disease progression while on ICB. Among these 7 patients with RNF43 mutations, 6 had additional mutations in APC or CTNNB1. Conclusions: Blood based genomic profiling can identify MSI-H cancers. Patients with MSI-H cancers resistant to ICB in this cohort have mutations in RNF43 as well as additional mutations in APC or CTNNB1, suggesting that co-activation of the WNT/B-Catenin pathway may be biologically important. Further study of the role of WNT/B-Catenin pathway activation in ICB resistance will be pursued using tumor tissue from this cohort.

3040 Background: Circulating tumor DNA next generation sequencing (ctDNA NGS) is increasingly being used to detect mutations (MT) in patients (pts) with metastatic (m) NSCLC. Limited data exist on the correlation of baseline ctDNA NGS profile and serial ctDNA NGS monitoring to response to immunotherapy. Methods: We conducted a prospective study in pts with mNSCLC receiving pembrolizumab monotherapy. Plasma was collected at weeks 0 (T0), 9 (T1), and 18 (T2). ctDNA NGS was performed using a 73 gene panel. Number of MTs and variant allelic fraction (VAF) were determined at baseline, and serially; change in mean VAF was calculated between T1-T0, and T2-T0. Response rate (RR) was assessed using RECIST 1.1. Correlations were made for pt characteristics, RR, progression free survival (PFS), and overall survival (OS). Results: We analyzed 95 samples from 33 pts, 21 female, median age 69 (range 51-89 years), smokers (n = 29), adenocarcinoma (n = 23), 25 pts enrolled at initial diagnosis, majority had high PD-L1 ≥50% (n = 29, 88%). At T0, 32 pts had detectable MT, median number of MTs was 4 (range 0-21), (non-synonymous MT = 3), most common MT was TP53 (n = 21). Confirmed PR was 27% (n = 9), clinical benefit rate (SD+PR) was 64% (n = 21), and 2 pts were not evaluable for response. Smokers were more likely to have higher number of MT at T0 (4 vs. 1 p = 0.003); there was no correlation with smoking and overall RR (p = 0.17). RR was not related to number of MT at T0, p = 0.37. A decrease in ctDNA VAF was seen in 6/9 pts with PR (mean VAF change range -0.11 to -0.001); 2/5 pts with PD showed an increase in mean VAF while 3 showed decrease. At median follow up of 9.26 months (mos), median PFS and OS were 7.4 and 10.5 mos, respectively. Median PFS was longer for pts with a decrease in ctDNA VAF at both T1-T0 (8.9 vs. 5 mos, p = 0.02) and T2-T0 (9.1 vs. 5.5 mos, p = 0.006). OS and additional biomarker analyses including correlation of response to a 2mb ctDNA plasma-based NGS panel will be reported at the meeting. Conclusions: Our results demonstrate that it is feasible to serially monitor plasma NGS, decline in mean ctDNA VAF correlates with radiographic response and PFS on immunotherapy with pembrolizumab.

Although the rules governing police interviews with suspects in custody have been well regulated since the introduction of the Police and Criminal Evidence Act 1984 (PACE) in England and Wales and Northern Ireland, concern has been expressed over the years about the vulnerable position of young suspects within the PACE regime and about the adequacy of the safeguards provided. This paper discusses the nature of police interviews with young people in Northern Ireland, drawing on the findings of a recent research project. The paper focuses particularly on the roles of the various participants (the interviewing officer(s), the appropriate adult and the solicitor) within the dynamics of advising young persons before and during the police interview and calls for a reconstruction of the role of the appropriate adult.

In the recent White Paper, Justice for All, the Government announced its intention to implement many of the measures recommended by the Criminal Courts Review completed by Lord Justice Auld in 20011 and declared its commitment to shifting the balance in the criminal justice system 'in favour of the victim and the community so as to reduce crime and bring more offenders to justice.'2 The Auld Review and the White Paper propose a range of reforms to nearly every stage of the criminal process. Many of these proposals have been included in the Criminal Justice Bill2002 which was introduced in the House of Commons on 21 St November 2002. These reforms, if implemented in full, would dramatically change criminal justice as we currently know it. These changes would impact upon every person that comes in contact with this process, not least those called upon to provide one of the most vital commodities in the process: evidence. Witnesses, who according to some are treated merely as 'information fodder'3, are vital to the criminal process and their treatment within the system has important implications, not only for prosecutions and convictions but also for the increasingly significant factor of public confidence in the criminal justice system. In her recent monograph, The Adversarial Process and the Vulnerable Witness, Louise Ellison claims that the current approach of both the courts and legislature to the treatment of the most vulnerable witnesses in our adversarial system leaves a lot to be desired. Ellison brings together an impressive body of research findings from a variety of academic, government and voluntary bodies across the world, and analyses the approach of the English criminal justice system to vulnerable and intimidated witnesses, paying particular attention to the most recent measures introduced under the Youth Justice and Criminal Evidence Act 1999. The limited approach in this jurisdiction of merely 'accommodating' vulnerable and intimidated witnesses within the traditional adversarial model is inadequate, she argues, both in securing the best evidence from these witnesses and in reducing the

Traditional accounts of fear of crime and victimisation have largely concentrated on factors associated with the individual such as their age, sex or ethnicity and more recently in Northern Ireland their religion. This conventional approach, adopted by many national victimisation surveys such as the British Crime Surveys and the Northern Ireland Crime Survey, has lead to assertions that, for example, women and older people are more anxious about victimisation and Protestants in Northern Ireland are more worried about terrorist attack than their Catholic counterparts. Drawing on the results of the first community based crime survey conducted in Northern Ireland, this paper explores the extent to which the community in which an individual lives, as opposed to their individual characteristics, directly impacts on their fear of crime. It is argued that, by overlooking the significance of community based factors, some criminologists may actually be obscuring our understanding of the dynamics of this fear.

We construct a birth and death process for the number of T cells belonging to one clonotype. Cells are released from the thymus into the peripheral lymphoid organs. We assume that after this time, no more T cells of this clonotype are exported by the thymus, so that further T cells of this clonotype can only be generated by homeostatic proliferation, when a T cell receives a survival signal and undergoes a single round of cell division. We show that eventual extinction is guaranteed. The late-time behaviour of the process before extinction takes place is described by the limiting conditional probability distribution (LCD), which we prove exists. We show how approximations are related to the LCD of the original process and use them to study the LCD in two special cases.

Recent advances in biomaterial surface engineering have shown that surface biomechanical, spatial and topographical properties can elicit control over fundamental biological processes such as cell shape, proliferation, differentiation and apoptosis. Along these lines, we have very recently shown that the self-assembly of block copolymers into thin films can be used as an extremely labile method to precisely position cellular adhesion molecules, at nanometre lateral spacings, to effect control over cell attachment and morphology. Here, we extend our work in 2-dimensional block copolymer films into the production of 3-dimensional porous block copolymer scaffolds. The reported method combines macro-scale temperature induced phase separation and micro-phase separation of block copolymers to produce highly porous scaffolds with surfaces comprised of nano-scale self-assembled block copolymer domains, representing a significant advance in currently available scaffold engineering technologies. The phase behaviour of these polymer-solvent systems is described and potential mechanisms leading to the observed structure formation are presented. The nano-domains have thereafter been functionalised with CGRGDS peptides throughout the scaffold and shown to effect changes in cell attachment and spreading, in agreement with previous 2-dimensional studies. These multi-scale, functional scaffolds are easy to manufacture and scaleable, making them ideal candidates for tissue engineering applications.