Tissue Next-generation Sequencing Research Articles

188 (PB068) - Updated Findings and Biomarker Analysis From the Ongoing Phase 1 Study of Enhancer of Zeste Homolog 2 (EZH2) Inhibitor CPI-0209 in Patients With Advanced Solid Tumors

188 (PB068) - Updated Findings and Biomarker Analysis From the Ongoing Phase 1 Study of Enhancer of Zeste Homolog 2 (EZH2) Inhibitor CPI-0209 in Patients With Advanced Solid Tumors

384P Concordance of genomic alterations in tumour tissue and ctDNA in patients with colorectal cancer referred for early phase trials

Plasma-based circulating tumour DNA (ctDNA) provides a minimally invasive, contemporaneous assessment of genomic alterations (GAs) and captures tumour heterogeneity. We compare GAs identified in ctDNA and archival tissue in patients (pts) with metastatic colorectal cancer (CRC) recruited to the Tumour Characterisation to Guide Experimental Targeted Therapy (TARGET) trial, aiming to match pts to early-phase cancer clinical trials (EPCCT). Archival tumour tissue was analysed using next generation sequencing (NGS) panels (24 genes), and plasma-based ctDNA was analysed using a local research NGS panel (641 genes). Results were discussed in a molecular tumour board (MTB) to define actionability. Factors associated with the likelihood of having additional GAs in ctDNA were explored using logistic regression analysis and odds ratio (OR). 101 pts with CRC were recruited to TARGET. The median age was 56 years and 61% were male. Pts had NGS testing panels from primary (70%) or metastatic (30%) archival tumour tissue. The median age of archival tissue tested was 25 months (range 0-117 months) and 98% of pts received active treatment in the intervening time. Tissue and ctDNA NGS identified clinically relevant GAs in 90% and 79% of pts respectively, with a median of 2 GAs per pt. The most common GAs in ctDNA were TP53 (53%), KRAS (47%), APC (44%), and PIK3CA (12%). There was poor concordance of identified GAs between tissue and ctDNA (agreement=66.3%, Cohen's k<0, p=0.91). 23% of pts had clinically relevant GAs in ctDNA that were not identified in tissue, the commonest being KRAS (29%). There was no association between the presence of additional GAs in ctDNA with number of treatment lines (OR:1.23, p= 0.32), age of archival tissue (OR: 1.01, p= 0.91) or primary vs. metastatic archival tissue site (OR: 0.34, p= 0.11). The presence of liver metastases was associated with a higher likelihood of concordance between tissue and ctDNA NGS (OR: 3.5, p= 0.03). Overall, ctDNA-derived GAs were considered actionable in 51% of pts. Of those, 28% were a potential match for an EPCCT. Plasma-based ctDNA can be used as a contemporaneous tool to identify actionable GAs and guide treatment options for pts with metastatic CRC.

Prevalence of ARID1A Mutations in Cell-Free Circulating Tumor DNA in a Cohort of 71,301 Patients and Association with Driver Co-Alterations.

Simple SummaryARID1A abnormalities disturb gene coding processes and correlate with immunotherapy responsiveness. We report the first blood sample-based genomic sequencing of ARID1A in DNA shed from tumors into the circulation (known as cell-free DNA (cfDNA) from liquid biopsy). Altogether, of 62,851 cancer patients with ≥1 cfDNA alteration in the blood, 3137 (5%) had ≥1 deleterious ARID1A alteration (a frequency similar to the ~6% generally reported in tissue sequencing), suggesting this non-invasive test’s value in detecting ARID1A. ARID1A alterations were most frequent in endometrial (21.3% of patients) and bladder cancer (12.9% of patients). As compared to blood samples without ARID1A aberrations, those with a functional (deleterious) ARID1A abnormality had more DNA alterations/sample (median, 6 versus 4; p < 0.0001) and more frequent co-alterations in one or more genes in key pathways promoting cancer development/progression, which may inform therapeutic strategies.ARID1A abnormalities disturb transcriptional processes regulated by chromatin remodeling and correlate with immunotherapy responsiveness. We report the first blood-based cell-free DNA (cfDNA) next-generation sequencing (NGS) ARID1A analysis. From November 2016 through August 2019, 71,301 patients with advanced solid tumors underwent clinical blood-derived cfDNA testing. Of these patients, 62,851 (88%) had ≥1 cfDNA alteration, and 3137 (of the 62,851) (5%) had ≥1 deleterious ARID1A alteration (a frequency similar to the ~6% generally reported in tissue NGS), suggesting this non-invasive test’s value in interrogating ARID1A. ARID1A cfDNA alterations were most frequent in endometrial cancer, 21.3% of patients; bladder cancer, 12.9%; gastric cancer, 11%; cholangiocarcinoma, 10.9%; and hepatocellular carcinoma, 10.6%. Blood samples with a functional ARID1A abnormality had more alterations/sample (median, 6 versus 4; p < 0.0001) and more frequent co-alterations in ≥1 gene in key oncogenic pathways: signal transduction, RAS/RAF/MAPK, PI3K/Akt/mTor, and the cell cycle. Taken together, our data suggest that liquid (blood) biopsies identify ARID1A alterations at a frequency similar to that found in primary tumor material. Furthermore, co-alterations in key pathways, some of which are pharmacologically tractable, occurred more frequently in samples with functional (deleterious) ARID1A alterations than in those without such aberrations, which may inform therapeutic strategies.

PRECISION ONCOLOGY- BASIC TO BEDSIDE

Basic science technologies and platforms have unraveled the molecular basis of carcinogenesis, both genetic and epigenetic events. Few of the events which are repeatedly observed and are actionable, gave birth to the science “Precision Oncology”. The evolution of molecular testing has made a major stride in the recent times, and has empowered personalized cancer medicine over the conventional blanket treatment. Among the important diagnostic technologies, Next Generation Sequencing (NGS) based in-vitro diagnostics have contributed substantially to comprehensive genomic profiling (CGP) in clinical practice that is meant for the detection of substitutions, insertions and deletions (indels), copy number alterations, and gene rearrangements, as well as genomic signatures including Microsatellite Instability (MSI), Loss of Heterozygosity (LOH), Homologous Recombination Defect (HRD Score), Tumor Mutation Burden (TMB). CGP has come into clinical practice with various guidelines endorsing its clinical utility in various indications, both tumor specific and tumor agnostic. Lung cancer is an excellent example where the role of NGS based comprehensive genomic profiling (CGP) has been recommended upfront either on tissue or blood (Liquid Biopsy). In ovarian cancer, calculation like LOH or HRD score, based on the tissue NGS, has widen the eligibility of PARP inhibitor drugs. Further, NTRK fusions, MSI and TMB biomarkers are universal supporting tumor agnostic approach. CGP is a tremendous diagnostic application but brings challenges especially interpretation which are affected by intra/inter tumor heterogeneity, identification of multiple driver mutations, multiple targets and treatment options, cross talk of various mutations and genomic signatures, and various signaling pathways, variable variant allele frequency (VAF), cut-offs for various scores like TMB, LOH, HRD. All these challenges are currently overcome by Molecular Tumor Boards, where basic science understanding in conjunct with clinical experience, has made a huge difference. Thus, offering true precision oncology with unique treatment options for individual patient.&nbsp;

Optimising CT-guided biopsies of sclerotic bone lesions in cancer patients

ObjectivesInvestigate the laboratory, imaging and procedural factors that are associated with a tumour-positive and/or NGS-feasible CT-guided sclerotic bone lesion biopsy result in cancer patients.MethodsIn total, 113 CT-guided bone biopsies performed in cancer patients by an interventional radiologist in one institution were retrospectively reviewed. Sixty-five sclerotic bone biopsies were eventually included and routine blood parameters and tumour marker levels were recorded. Non-contrast (NC) biopsy CTs (65), contrast-enhanced CTs (24), and PET/CTs (22) performed within four weeks of biopsy were reviewed; lesion location, diameter, lesion-to-cortex distance, and NC-CT appearance (dense-sclerosis versus mild-sclerosis) were noted. Mean NC-CT, CE-CT HU, and PET SUVmax were derived from biopsy tract and lesion segmentations. Needle diameter, tract length, and number of samples were noted. Comparisons between tumour-positive/negative and next-generation sequencing (NGS)-feasible/non-feasible biopsies determined significant (p < 0.05) laboratory, imaging, and procedural parameter differences.ResultsSeventy-four percent of biopsies were tumour-positive. NGS was feasible in 22/30 prostate cancer patients (73%). Neither laboratory blood parameters, PET/CT availability, size, nor lesion-to-cortex distance affected diagnostic yield or NGS feasibility (p > 0.298). Eighty-seven percent of mildly sclerotic bone (mean 244 HU) biopsies were positive compared with 56% in dense sclerosis (622 HU, p = 0.005) and NC-CT lesion HU was significantly lower in positive biopsies (p = 0.003). A 610 HU threshold yielded 89% PPV for tumour-positive biopsies and a 370 HU threshold 94% PPV for NGS-feasible biopsies. FDG-PET and procedural parameters were non-significant factors (each p > 0.055).ConclusionIn cancer patients with sclerotic bone disease, targeting areas of predominantly mild sclerosis in lower CT-attenuation lesions can improve tumour tissue yield and NGS feasibility.Key Points• Areas of predominantly mild sclerosis should be preferred to areas of predominantly dense sclerosis for CT-guided bone biopsies in cancer patients.• Among sclerotic bone lesions in prostate cancer patients, lesions with a mean HU below 370 should be preferred as biopsy targets to improve NGS feasibility.• Laboratory parameters and procedure related factors may have little implications for CT-guided sclerotic bone biopsy success.

The Role of Liquid Biopsy in the Diagnostic Testing Algorithm for Advanced Lung Cancer

The discovery of therapeutically targetable oncogenic driver alterations has led to marked improvements in NSCLC outcomes. Targeted agents have been approved for an expanding list of biomarkers. Consequently, the accurate and timely identification of targetable alterations with diagnostic molecular profiling is crucial. The use of multiplexed tissue assays, such as next-generation sequencing (NGS), has increased significantly. However, significant limitations with tissue NGS remain, such as insufficient tissue, scheduling limitations, the need for repeat biopsies, and long turnaround times. Liquid biopsies, using plasma circulating tumor DNA (ctDNA), have the potential to overcome these issues, with simpler sample processing requirements, greater convenience, and better patient acceptability. In particular, an early liquid biopsy may allow patients access to highly effective therapies faster, allow better symptom control and quality of life, prevent rapid clinical deterioration, and reduce patient anxiety at diagnosis. More broadly, it may also allow for the more cost-effective delivery of healthcare to patients.

Audit of Molecular Mechanisms of Primary and Secondary Resistance to Various Generations of Tyrosine Kinase Inhibitors in Known Epidermal Growth Factor Receptor-Mutant Non-small Cell Lung Cancer Patients in a Tertiary Centre

AimsPresently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary). Materials and methodsThis was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent™ Oncomine™ Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit. ResultsThe results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation. ConclusionThis study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.

Evidence of neural crest cell origin of a DICER1 mutant CNS sarcoma in a child with DICER1 syndrome and NRAS-mutant neurocutaneous melanosis.

Evidence of neural crest cell origin of a DICER1 mutant CNS sarcoma in a child with DICER1 syndrome and NRAS-mutant neurocutaneous melanosis.

Abstract 3404: Detection of circulating tumor DNA using CRISPR-Cas13a in non-small cell lung cancer

Abstract Background: Lung cancer remains the leading cause of cancer-related death and despite advances in low-dose CT screening, most lung cancer patients still present with advanced disease. The development of assays that are sensitive, specific, economical, and non-invasive may lead to the development of further effective screening strategies. Non-invasive liquid biopsy by detection in plasma of circulating tumor DNA (ctDNA) carrying somatic lung cancer mutations is now routinely used for identifying targeted therapy options in advanced non-small cell lung cancer (NSCLC) patients. Recent adaptation of bacterial CRISPR-Cas13a systems with promiscuous ribonuclease activity into reporter assays for the sensitive and economical detection of specific nucleic acid sequences makes an attractive system for ctDNA detection without needing more costly next-generation sequencing (NGS) techniques. Methods: We adapted the CRISPR-Cas13a system used in the SHERLOCK detection assay for the detection of common driver mutations (KRAS G12C/V, EGFR Exon 19 deletion, and EGFR L858R) in NSCLC and tested the assay on a retrospective cohort of 52 NSCLC patients with known mutations (based on tissue NGS) and with plasma obtained prior to or at the time of any surgical resection and a prospective cohort of 71 patients who underwent lung resection with plasma collected at the time of surgical resection. Results: We designed several CRISPR guide RNAs targeting common EGFR and KRAS oncogenic mutations in NSCLC and were able to generate sensitive CRISPR-Cas13a detection assays against KRAS G12C and G12V, EGFR L858R, and EGFR E746_A750 exon 19 deletion mutations that could identify allele fractions below 1% in reference standards. We then tested plasma of a 52 patient (33 advanced stage IIIB and IV, 19 resectable stage I-IIIA) retrospective cohort with known KRAS and EGFR mutations and matching non-cancer controls using the CRISPR-Cas13a assay as well as with NGS panels (ArcherDx). Using NGS, we were able to confirm ctDNA in 15 samples which we were also able to detect using the CRISPR-Cas13a assay with similar allele frequencies. The CRISPR-Cas13a assay was able to identify ctDNA in 42 out of the 52 retrospective NSCLC samples including 13 out of 19 samples from resectable-stage patients, although the detection rate decreased with earlier stage I and II patients who have a much lower ctDNA level in plasma. We then prospectively collected a cohort of 71 subjects who underwent surgical resection with 38 subjects with lung adenocarcinoma (mostly stage I and II), 11 subjects with benign nodules, and 22 subjects with a different histology malignancy. Our CRISPR-Cas13a assay was able to detect 9 out of 38 lung adenocarcinomas with no false positives in subjects with benign nodules. Conclusion: We adapted a CRISPR-Cas13a assay for the detection of ctDNA in NSCLC and evaluated this assay on plasma from NSCLC subjects. Citation Format: Edwin H. Yau, Bojidar Kandar, Catrina Ting, Sai Yendamuri, Mary Reid. Detection of circulating tumor DNA using CRISPR-Cas13a in non-small cell lung cancer [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 3404.

Abstract 3411: Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients

Abstract Background: The analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision making and disease monitoring. A suitable diagnostic-grade platform is required for detection of tumor-specific mutations with high sensitivity in circulating cell-free DNA (ccfDNA) of cancer patients. Methods: In an international multicenter study conducted by three laboratories participating in the CANCER-ID consortium, a cohort containing 177 cell-free plasma samples at baseline, follow-up or disease progression derived from 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC), relevant mutations were tested using the UltraSEEK® Lung Panel on the MassARRAY® System covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2 and PIK3CA, and compared against mutation-specific droplet digital PCR (ddPCR) and tumor tissue next-generation sequencing (NGS) performed in routine diagnostics. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy were analyzed. Results: The UltraSEEK® Lung Panel revealed an overall detection rate of 87.2% irrespective of ccfDNA input, and a specificity of &amp;gt;99.5% using the reference material. When comparing the 131 variants identified in the patient-derived cell-free plasma using UltraSEEK® with mutation-specific ddPCR analyses, a concordance of 90% was found. The 77 diagnostically or clinically relevant variants identified in 66 pretreatment tumor tissue showed an overall concordance with UltraSEEK® of 73% at baseline with a false-negative rate of 3%. When restricting to therapeutically targetable mutations, the concordance elevated to 85%. A decrease in ctDNA levels at 4-6 weeks after start of treatment detected with UltraSEEK® correlated with a prolonged median PFS (46 vs 11 weeks; P&amp;lt;0.05) and OS (145 vs 39 weeks; P&amp;lt;0.001). In four out of thirteen NSCLC patients treated with targeted therapy, resistance mutations were identified using UltraSEEK® at disease progression, implicating the requirement of a therapy switch. Conclusion: Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect both mutations at diagnosis and disease progression, and to monitor treatment response. Citation Format: Paul van der Leest, Melanie Janning, Naomi Rifaela, Maria L. Aguirre Azpurua, Jolanthe Kropidlowski, Sonja Loges, Nicholas Lozano, Alexander Sartori, Darryl Irwin, Pierre-Jean Lamy, T. Jeroen N. Hiltermann, Harry J. Groen, Klaus Pantel, Léon C. van Kempen, Harriet Wikman, Ed Schuuring. Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients [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 3411.

Plasma first: Accelerating lung cancer diagnosis through liquid biopsy.

3039 Background: Molecular profiling of tumor tissue is the gold standard for treatment decision making in advanced non-small cell lung cancer. Results may be delayed or unavailable due to insufficient tissue samples or prolonged wait times for biopsy, pathology assessment and testing. We piloted the use of plasma molecular testing as part of the initial diagnostic work-up for patients with suspected advanced lung cancer (NCT04863924). Methods: Patients with radiologic evidence of advanced lung cancer referred to the lung rapid diagnostic program underwent plasma circulating tumor DNA (ctDNA) testing using InVisionFirst-Lung, a next-generation sequencing (NGS) assay targeting 37 genes. Standard tissue testing was performed with comprehensive NGS (Oncomine). The primary endpoint was time to treatment in stage IV NSCLC patients compared to an historical pre-COVID-19 cohort (2018-9). Secondary endpoints included actionable targets identified in plasma, % of patients starting targeted therapy based on liquid biopsy and result turnaround time (TAT). Results: Between July 1 to December 31, 2021, 60 patients were enrolled. Median age was 70 years (range 33-91), 52% were female, 57% Caucasian, 48% never smokers. Of these, 73% had NSCLC, 12% small cell, 10% non-lung pathology and 5% declined tissue biopsy. Of 44 NSCLC patients, 5 (11%) had early-stage disease and underwent curative therapy. Most stage IV patients (79%) had systemic treatment. Median time to treatment initiation in the study cohort was 34 days (n = 31, range 10-90) versus 62 days (n = 101, range 13-159) in the historical cohort (p&lt;0.0001). Two thirds (N = 23) of stage IV NSCLC patients had actionable alterations identified, (30% in current/ex-smokers); 18 started targeted therapy including 10 based on plasma results before tissue results were available. Median TAT was 7 days for plasma from blood draw to reporting (range 4-14) and 26 days for tissue molecular testing (range 11-42), p&lt;0.0001. Concordance was high between plasma and tissue testing (70%). Liquid biopsy identified actionable alterations for 3 patients not identified by tissue NGS. In 4 cases, plasma testing failed to identify actionable alterations detected in tissue, due to undetectable plasma ctDNA. Conclusions: Liquid biopsy in the initial diagnostic workup of patients with suspected advanced NSCLC leads to faster molecular results and shortens time to treatment compared to tissue testing alone. Supplementing the current standard of tissue molecular testing with a plasma-first approach during the diagnostic work up of patients with suspected advanced lung cancer may increase access to precision medicine and improve patient outcomes. Clinical trial information: NCT04863924. [Table: see text]

Identification of an optimal circulating tumor DNA (ctDNA) shedding threshold to detect actionable driver mutations in colorectal and pancreatic adenocarcinoma.

3571 Background: In colorectal cancer (CRC), mutations in KRAS, NRAS, and BRAF predict resistance to anti-EGFR therapies. In pancreatic ductal adenocarcinoma (PDAC), ~90% of patients harbor KRAS mutations, while KRAS wild-type tumors often have clinically actionable fusion alterations. Thus, in both cancers, the accurate ascertainment of RAS and BRAF driver status is essential. Sequencing of cell-free DNA (cfDNA) from plasma allows convenient assessment of a tumor’s molecular profile, but sensitivity can be limited by low ctDNA shedding. We sought to establish a ctDNA shedding threshold at which actionable driver mutations can be reliably detected. Methods: Molecular reports and matched clinical data were obtained from the Duke Molecular Registry of Tumors and the SCRUM-Japan GOZILA and GI-SCREEN. CRC or PDAC patients with a pathogenic KRAS, NRAS, or BRAF activating point mutation (“driver”) present on tissue next-generation sequencing (NGS) assays and who also had cfDNA assay available were included. Tissue NGS included Foundation One CDx and Oncomine Comprehensive Assay. Guardant 360 (G360) was the sole plasma cfDNA assay. 131 CRC and 24 PDAC cases with 189 total G360 assays met criteria and were included. Samples were analyzed according to detection of the driver mutation and the maximum mutant allele frequency (MAF) of non-driver mutations on G360. An optimal cut-point for max MAF was explored among the CRC and PDAC patients using a maximally selected Wilcoxon rank statistic method. Results: 76.8% of driver mutations were in KRAS, 22.6% in BRAF, and 1.9% in NRAS with an overlap of 1 BRAF and 1 NRAS mutation with a KRAS mutation. Overall sensitivity of G360 for drivers was 83.0% for CRC and 54.2% for PDAC. No variants were detected on G360 in 9.1% of CRC and 37.5% of PDAC. Sensitivity for driver mutations increased with higher maximum non-driver MAF, with MAF &gt; 1% predicting sensitivity &gt; 98% (Table). Optimal cut-point analysis identified MAF of &gt; 0.34% (p &lt; 0.0001), above which the driver was identified in 97% of patients and below which only 27%. Conclusions: In our study, non-driver MAF &gt; 1% on cfDNA NGS predicts high sensitivity for RAS and BRAF mutations and thus is adequate to guide clinical decisions such as anti-EGFR therapy in CRC, evaluation for fusions in PDAC, and validity in clinical trials. MAF ≤0.34% is a clear threshold to consider an assay inadequate and thus seek alternative testing. Sensitivity rises for MAF between 0.35 and 1% but will require greater patient numbers to establish clinically relevant sensitivity thresholds. These results will be updated with additional data for final presentation. [Table: see text]

Evaluation of tumor gene expression profile (GEP) in minority patients with stage IV non-small cell lung cancer (NSCLC).

e21043 Background: Differences in tumor gene expression profile (GEP) may contribute to disparities in lung cancer incidence and survival between racial and ethnic groups. For patients with stage IV NSCLC, tumor biomarker analysis is important for identification of potentially effective targeted therapies. However, despite advancements in biomarker testing and targeted therapy, minority patient enrollment in clinical trials remains low and it has been shown that minority patients are less likely to undergo next-generation sequencing (NGS) compared to Non-Hispanic Whites (NHW). As such, frequencies of tumor biomarkers in minority populations in the United States are not well-defined. This study aimed to determine the extent of tumor GEP diversity in minority patients vs NHW. Methods: This retrospective study included 196 adult patients with stage IV NSCLC that had a baseline NGS of tumor tissue or blood (liquid biopsy) performed between July 1, 2015 and June 30, 2021. Minority populations included Hispanic, African American, and Asian patients. Data collected utilizing the electronic medical record included patient demographics, detection of tumor biomarkers and treatment initiated. Descriptive statistics were calculated for all demographic and clinical variables. Comparisons of actionable epidermal growth factor receptor (EGFR) mutation frequency between NHW and minority patients were done using Chi-squared and Fisher’s exact tests. Results: Of the 196 patients evaluated, baseline characteristics included 46% male, 67% former smokers, 54% NHW, 31% Hispanic, 9% African American, 5% Asian, and 1% other. The median age at diagnosis was 66 ± 12 years. Actionable mutations (EGFR, KRAS, BRAF, ROS-1, ALK, MET, NTRK 1-3) were found in 50% of patients (see Table). The frequency of actionable EGFR mutations was higher in minority patients overall compared to NHW (39.3% vs 21.5%; p &lt; 0.01). The frequency of actionable EGFR mutations was similar between NHW and Hispanic patients (21.5% vs 27.9%; p = 0.35), however differences were observed comparing NHW to African American (21.5% vs 50%; p = 0.02) and Asian (21.5% vs 90%; p &lt; 0.01) patients, respectively. Conclusions: Among patients with stage IV NSCLC, the GEP of minority patients varied from that of NHW. Asian patients had the highest frequency of EGFR mutations, followed by African American and Hispanic patients, respectively. Larger studies are needed to validate these findings.[Table: see text]

Longitudinal Shifts of Solid Tumor and Liquid Biopsy Sequencing Concordance in Metastatic Breast Cancer.

PURPOSETissue-based next-generation sequencing (NGS) in metastatic breast cancer (mBC) is limited by the inability to noninvasively track tumor evolution. Cell-free DNA (cfDNA) NGS has made sequential testing feasible; however, the relationship between cfDNA and tissue-based testing in mBC is not well understood. Here, we evaluate concordance between tissue and cfDNA NGS relative to cfDNA sampling frequency in a large, clinically annotated mBC data set.METHODSTempus LENS was used to analyze deidentified records of mBC cases with Tempus xT (tissue) and xF (cfDNA) sequencing results. Then, various metrics of concordance were assessed within overlapping probe regions of the tissue and cfDNA assays (104 genes), focusing on pathogenic variants. Variant allele frequencies of discordant and concordant pathogenic variants were also compared. Analyses were stratified by mBC subtype and time between tests.RESULTSRecords from 300 paired tissue and liquid biopsies were analyzed. Median time between tissue and blood collection was 78.5 days (standard deviation = 642.99). The median number of pathogenic variants/patient was one for cfDNA and two for tissue. Across the cohort, 77.8% of pathogenic tissue variants were found in cfDNA and 75.7% of pathogenic cfDNA variants were found in tissue when tests were ≤ 7 days apart, which decreased to 50.3% and 51.8%, respectively, for > 365 days. Furthermore, the median patient-level variant concordance was 67% when tests were ≤7 days apart and 30%-37% when > 30 days. The median variant allele frequencies of discordant variants were generally lower than those of concordant variants within the same time frame.CONCLUSIONWe observed high concordances between tissue and cfDNA results that generally decreased with longer durations between tests. Thus, cfDNA NGS reliably measures tissue genomics and is likely beneficial for longitudinal monitoring of molecular changes in mBC.

Primary results from JUPITER, a phase 2 basket trial of combination therapy with trastuzumab and pertuzumab in patients with HER2-amplified solid tumors.

3131 Background: Human epidermal growth factor receptor 2 (HER2) gene amplification or mutations have emerged as oncogenic drivers and therapeutic targets not limited to breast and gastric cancers, but also in a variety of cancers. Despite its considerable therapeutic potential, the evidence has not been established. To address this unmet need, we conducted an organ-agnostic basket trial targeting HER2-amplified solid tumors. Methods: JUPITER is a multicenter, single-arm, phase 2 basket trial for solid tumor patients (pts) with HER2 amplification determined by next-generation sequencing (NGS). Both tissue and liquid NGS results were allowed. HER2 amplification by ISH or HER2 overexpression by IHC were not used for inclusion. Pts had treatment-refractory metastatic tumors, or rare cancers without established standard of care. Breast, gastric, and colorectal cancers were excluded. Pts were treated with intravenous trastuzumab (8 mg/kg loading dose followed by 6 mg/kg) and pertuzumab (840 mg loading dose followed by 420 mg) every 3 weeks until disease progression or any other reason for discontinuation. Tumor response was assessed using RECIST v1.1. Primary endpoint was ORR by blinded independent central review (BICR), and secondary endpoints were ORR assessed by the investigators, progression-free survival (PFS), overall survival (OS), duration of response (DOR), and safety. We set the ORR threshold 5% and expected ORR was 20%. Estimated sample size was 38 patients with one-sided alpha 2.5% and power 80%. Results: Between April 2019 and June 2020, 42 pts were consented, and 40 pts were treated. Median age was 62 (range, 21-86) and 60% were females. The most common diagnosis was biliary tract cancer (20%), followed by salivary ductal carcinoma (12.5%) and endometrial cancer (12.5%). At data cutoff (1 Sep 2021), ORR by BICR was 22.5% (95%CI: 10.8%-38.5%). ORR assessed by the investigator was 25% (95%CI: 12.7%-41.2%). PFS, OS and DOR were not reached at data cutoff; 3 responders remained on treatment. Of 40 pts, 32.5% had grade ≥ 3 adverse events; 10% were treatment-related, including neutropenia, hypertension, peripheral sensory neuropathy and lymphoedema (all grade 3). No treatment-related death was observed. Exploratory biomarker analysis of response and resistance is in progress. Conclusions: Combination therapy with trastuzumab and pertuzumab was well tolerated and showed promising efficacy for the patients with HER2-amplified solid tumors determined by NGS. Clinical trial information: jRCT2031180150.

Exhaled Breath Condensate (EBC) analysis of circulating tumour DNA (ctDNA) using a lung cancer specific UltraSEEK oncogene panel

IntroductionSmall diagnostic tissue samples can be inadequate in testing an expanding list of validated oncogenic driver alterations and fail to reflect intratumour heterogeneity (ITGH) in lung cancer. Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows promise as a lung-specific liquid biopsy. MethodsIn this prospective, proof-of-concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples from 125 patients with lung cancer and compared results to plasma and EBC for 5 oncogenic driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK™ Lung Panel on the MassARRAY® System, Agena Bioscience, San Diego, CA, USA). ResultsThere was a significantly higher failure rate due to unamplifiable DNA in tissue NGS (57/125, 45.6%) compared to plasma (27/125, 21.6%, p < 0.001 and EBC (26/125,20.8%, p ≤ 0.001. Consequently, both plasma and EBC identified higher number of mutations compared to tissue NGS. Specifically, there were significantly higher numbers of mutations detected in EGFR, KRAS and PIK3CA in plasma (p = 9.82 × 10−3, p = 3.14 × 10−5, p = 1.95 × 10−3) and EBC (p = 2.18 × 10−3, p = 2.28 × 10−4,p = 0.016) compared to tissue NGS. There was considerable divergence in mutation profiles between plasma and EBC with 34/76 (44%) mutations detected in plasma and 37/74 (41.89%) in EBC unique to their respective liquid biopsy. ConclusionsThe results suggest that EBC is effective in identifying clinically relevant alterations in patients with lung cancer using UltraSEEK™ and has a potential role as an adjunct to plasma testing.

PD46-04 BRCA1/2 AND ATM MUTATED METASTATIC PROSTATE CANCERS MAY PRESENT WITH LOW SERUM PSA

You have accessJournal of UrologyCME1 May 2022PD46-04 BRCA1/2 AND ATM MUTATED METASTATIC PROSTATE CANCERS MAY PRESENT WITH LOW SERUM PSA Hyunho Han, Cheol Keun Park, Nam Hoon Cho, Jong Su Lee, Won Sik Ham, Woong Kyu Han, and Young Deuk Choi Hyunho HanHyunho Han More articles by this author , Cheol Keun ParkCheol Keun Park More articles by this author , Nam Hoon ChoNam Hoon Cho More articles by this author , Jong Su LeeJong Su Lee More articles by this author , Won Sik HamWon Sik Ham More articles by this author , Woong Kyu HanWoong Kyu Han More articles by this author , and Young Deuk ChoiYoung Deuk Choi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002614.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: PARP inhibitors have shown survival benefits in M-CRPC patients with homologous recombination repair (HRR) genes (BRCA1/2, and ATM) mutations. Germline panel test of the HRR genes and tumor tissue next generation sequencing (NGS) are recommended in men with metastatic prostate cancer. The reported incidences of BRCA1,2 and ATM gene mutations are 5-10%, yet it is unclear whether the mutations are enriched in advanced cancer and associated with clinical features. We assessed the incidences and clinical characteristics of HRR genes BRCA1/2 and ATM mutated prostate cancer. METHODS: Our institute has incorporated prostate cancer tissue NGS in clinical practice since Jun 2019. Indications include prostate cancer of high risk (GGG4-5, cT3/4, N1 or M1). Trusight oncology 170/500 panels were used. We assessed the incidences of BRCA1/2 and ATM mutations (including variant of unknown significance) in the tested population. Clinical characteristics of HRR mutated tumors and wild-type tumors were compared. T-test (two-sided) with Welch’s correction was used. RESULTS: We evaluated 126 consecutive prostate cancer tissue NGS data (from Jun 2019 and April 2021). At the time of diagnosis, cT3/4, N1 and M1 stages were 107(85%), 54(43%) and 35(28%) samples, respectively. BRCA1/2 and ATM mutation frequencies were similar across the stages: 32% at T2 vs 34% at T3/4; 35% at N0 vs 31% at N1; 32% at M0 vs 37% at M1. HRR mutations tended to be more frequent in GGG 5 than GGG1-4 (41% vs 28%, p = 0.15). Of metastatic subgroup (N1 or M1, n=62), mean PSA at the time of diagnosis was significantly lower in HRR mutated tumors than wild-type (82ng/mL vs 561ng/mL, p = 0.02). Similar trend was noted in a M-CRPC public dataset (188ng/mL vs 76ng/mL, p = 0.09) (Abida et al., PNAS, 2019). In localized subgroup (N0 and M0, n=64), PSA was not significantly different (58ng/mL vs 26ng/mL, p=0.30). CONCLUSIONS: BRCA1/2 and ATM mutated metastatic prostate cancers may present with low serum PSA. Source of Funding: NA © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e789 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hyunho Han More articles by this author Cheol Keun Park More articles by this author Nam Hoon Cho More articles by this author Jong Su Lee More articles by this author Won Sik Ham More articles by this author Woong Kyu Han More articles by this author Young Deuk Choi More articles by this author Expand All Advertisement PDF DownloadLoading ...

Concordance analysis between liquid biopsy (ctDNA) and tumor DNA molecular profiles from panel-based next-generation sequencing

IntroductionAnalysis of circulating tumor DNA (ctDNA), also known as liquid biopsy, has been postulated to be a useful test in the prognostication, molecular profiling, and monitoring of cancer patients. In this series we aimed to analyze the concordance between the mutation status of formalin-fixed paraffin-embedded (FFPE) tumor samples and matched ctDNA, considering tumor molecular profiling as the gold standard technique. MethodsThis retrospective study included cancer patients with complete diagnostics and gene mutations detected in a previous FFPE tumor tissue Next-Generation Sequencing (NGS) study with a matched frozen plasma sample available for an NGS ctDNA assay. Results and discussionSixty patients were included, 24 with colorectal carcinoma (CRC) and 36 with non-small cell lung cancer (NSCLC). In 27.1% of ctDNA studies a new mutation not previously detected in the matched tumor was found. 11.9% of these ctDNA results had the potential to impact clinical management. Globally, the concordance rate between FFPE tumor samples and ctDNA was 44.4%. When tumors were stratified by stage, the concordance was 76.5%, 70%, 36.4%, and 0% in tumor stages IV, III, II, and I, respectively. ctDNA molecular profiles showed a good concordance rate in advanced stage tumors and identified undetected mutations in tumor tissues. In early tumor stages the concordance was low, casting doubt on the usefulness of ctDNA in these patients.

Epidermal Growth Factor Receptor Inhibition in Epidermal Growth Factor Receptor-Amplified Gastroesophageal Cancer: Retrospective Global Experience.

Subset analyses from phase III evaluation of epidermal growth factor receptor inhibition (EGFRi) suggest improved outcomes in patients with EGFR-amplified gastroesophageal adenocarcinoma (GEA), but large-scale analyses are lacking. This multi-institutional analysis sought to determine the role of EGFRi in the largest cohort of patients with EGFR-amplified GEA to date. A total of 60 patients from 15 tertiary cancer centers in six countries met the inclusion criteria. These criteria required histologically confirmed GEA in the metastatic or unresectable setting with EGFR amplification identified by using a Clinical Laboratory Improvement Amendments-approved assay, and who received on- or off-protocol EGFRi. Testing could be by tissue next-generation sequencing, plasma circulating tumor DNA next-generation sequencing, and/or fluorescence in situ hybridization performed by a Clinical Laboratory Improvement Amendments approved laboratory. Treatment patterns and outcomes analysis was also performed using a deidentified clinicogenomic database (CGDB). Sixty patients with EGFR-amplified GEA received EGFRi, including 31 of 60 patients (52%) with concurrent chemotherapy. Across treatment lines, patients achieved a 43% objective response rate with a median progression-free survival of 4.6 months (95% CI, 3.5 to 6.4). Patients receiving EGFRi in first-, second-, and third-line therapy achieved a median overall survival of 20.6 months (95% CI, 13.5 to not reached [NR]), 9 months (95% CI, 7.9 to NR), and 8.4 months (7.6 to NR), respectively. This survival far exceeded the 11.2-month (95% CI, 8.7 to 14.2) median overall survival from first-line initiation of non-EGFRi therapy in patients with EGFR-amplified GEA in the CGDB. Despite this benefit, analysis of the CGDB (January 2011-December 2020) suggests that only 5% of patients with EGFR-amplified GEA received EGFRi. Patients with EGFR-amplified GEA derive significant benefit from EGFRi. Further prospective investigation of EGFRi in a well-selected patient population is ongoing in an upcoming trial of amivantamab in EGFR and/or MET amplified GEA.

Plasma Genotyping at the Time of Diagnostic Tissue Biopsy Decreases Time-to-Treatment in Patients With Advanced NSCLC—Results From a Prospective Pilot Study

IntroductionThe availability of targeted therapies has transformed the management of advanced NSCLC; however, most patients do not undergo guideline-recommended tumor genotyping. The impact of plasma-based next-generation sequencing (NGS) performed simultaneously with diagnostic biopsy in suspected advanced NSCLC has largely been unexplored. MethodsWe performed a prospective cohort study of patients with suspected advanced lung cancer on the basis of cross-sectional imaging results. Blood from the time of biopsy was sequenced using a commercially available 74-gene panel. The primary outcome measure was time to first-line systemic treatment compared with a retrospective cohort of consecutive patients with advanced NSCLC with reflex tissue NGS. ResultsWe analyzed the NGS results from 110 patients with newly diagnosed advanced NSCLC: cohorts 1 and 2 included 55 patients each and were well balanced regarding baseline demographics. In cohort 1, plasma NGS identified therapeutically informative driver mutations in 32 patients (58%) (13 KRAS [five KRAS G12C], 13 EGFR, two ERRB2, two MET, one BRAF, one RET). The NGS results were available before the first oncology visit in 85% of cohort 1 versus 9% in cohort 2 (p < 0.0001), with more cohort 1 patients receiving a guideline-concordant treatment recommendation at this visit (74% versus 46%, p = 0.005). Time-to-treatment was significantly shorter in cohort 1 compared with cohort 2 (12 versus 20 d, p = 0.003), with a shorter time-to-treatment in patients with specific driver mutations (10 versus 19 d, p = 0.001). ConclusionsPlasma-based NGS performed at the time of diagnostic biopsy in patients with suspected advanced NSCLC is associated with decreased time-to-treatment compared with usual care.