Insufficient Tumor Tissue Research Articles

Flushing fluid of post-bronchoscopic biopsy as a novel liquid biopsy specimen for genome profiling in advanced lung cancer.

e15038 Background: Tumour tissue is the primary substrate for molecular testing in advanced NSCLC. However, obtaining sufficient tissue samples from biopsy for molecular testing can often be challenging. Flushing fluid of post-bronchoscopic biopsy, which contains tumor-derived DNA, may serve as a supplement for genotyping. The aim of this study is to assess the efficacy and precision of detecting gene alterations in flushing fluid samples and evaluate their clinical potential. Methods: Flushing fluid, matched tissue (TIS), and whole blood were prospectively collected from 25 patients with stage III-IV NSCLC between June 2022 and December 2023. Ten milliliters of flushing fluid and whole blood were used to prepare flushing fluid supernatant (FFS) and plasma (PLA), respectively. Capture-based targeted sequencing of TIS, FFS and PLA samples was performed using a universal oncology panel. Results: In this cohort of 25 patients, there was a male predominance (22 out of 25) and the mean age was 60.2 years. The majority of patients were diagnosed with lung adenocarcinoma (23 out of 25), and smokers outnumbered non-smokers (16 out of 25). Furthermore, 18 out of 25 patients presented with metastatic disease at diagnosis. The performance of three sample types regarding key quality control indices was evaluated first. FFS exhibited a maximum allelic fraction similar to TIS, while PLA showed a significantly lower than TIS and FFS. Subsequently, we compared the positive detection rates, with TIS, FFS, and PLA achieving respective rates of 100%, 96%, and 80%. A total of 495 mutation events were detected from the three samples (FFP:209, FFS:196, PLA:90), Among these, 161 were concordant between TIS and FFS samples, and 72 were concordant between TIS and PLA samples. The sensitivity and positive predictive value (PPV) of detecting eight established driver alterations in NSCLC were compared using TIS as the gold standard with the two liquid biopsies. FFS demonstrated superior sensitivity and PPV compared to PLA (80.5% vs. 56.1%, 97% vs. 88.5%) and showed equivalent performance for EGFR mutation. Furthermore, estimates of tumor mutational burden (TMB) based on TIS and FFS were highly correlated (Pearson r= 0.97). Conclusions: Our study demonstrated that FFS performs comparably to matched TIS and superiorly to PLA for genotyping advanced NSCLC. FFS emerges as a valuable specimen source, especially in patients with insufficient tumor tissue.

Biomarker testing in early-stage NSCLC: Results from the MYLUNG Consortium.

8047 Background: The Molecularly Informed Lung Cancer Treatment in a Community Cancer Network: A Pragmatic Consortium (MYLUNG) program seeks to identify and resolve barriers to biomarker testing in patients (pts) with non-small-cell lung cancer (NSCLC). We previously reported prospectively collected molecular testing data in pts with Stage IB-IV NSCLC (Evangelist et al. 2023). Herein we report additional data on molecular testing rates and patterns in the cohort of pts with early-stage (ES) NSCLC. Methods: Pts were enrolled from 12/20-9/22 at 18 community practices (76 sites) across the US Oncology Network. Analyses performed in the ES cohort include rates of molecular testing for targets with approved (PD-L1 , EGFR) or emerging ( ALK) therapies for ES NSCLC and next-generation sequencing (NGS); reasons for not testing; and treatment (Tx) received, including targeted therapy and immunotherapy (IO). Analyses are preliminary as data collection continues over 5-year follow-up. Results: Included in this analysis were 284 pts with newly diagnosed Stage IB-IIIA NSCLC: median age 68y (range 32, 92); 50% female; 54% ECOG 0-1; 61% adenocarcinoma, 36% squamous cell carcinoma. In total, 76% of pts (82% of pts with adenocarcinoma vs 67% with squamous cell carcinoma) had molecular testing ordered prior to or within 12 weeks of initiating first systemic therapy. Rates of testing (either alone or inclusive of other tests) were PD-L1 ,74%; EGFR,71%; ALK, 62%; EGFR + PD-L1, 61%. In total, 52% of pts had NGS testing; overall, 16% received no molecular testing. The most common reasons for not testing were patient and/or provider attitude/perception (34%) and insufficient tumor tissue (25%). Conclusions: New therapeutic options provide a catalyst to increase molecular testing in ES NSCLC. EGFR and PD-L1 testing are now standard for the management of these pts, with other biomarkers emerging. In this study, 54% of pts received testing for PD-L1, EGFR, and ALK, with additional therapy being offered in pts with biomarker-positive tumors; however, 16% still received no molecular testing at all. For pts with EGFRwt/PD-L1+ tumors, prescription of IO increased 1.8-fold post-adjuvant FDA approval. Data from this and future analyses will be used to implement interventions to improve molecular testing rates in NSCLC. [Table: see text]

Personalized circulating tumor (ct)DNA for monitoring disease status in HPV-negative head and neck squamous cell carcinoma.

6056 Background: Highly sensitive minimal residual disease (MRD) assays using circulating tumor (ct)DNA have broad clinical potential and are impacting cancer care. Circulating human papillomavirus (HPV) DNA has emerged as a biomarker of occult MRD among patients (pts) with HPV+ oropharyngeal cancers, but most head and neck squamous cell carcinomas (HNSCC) are not virally driven. Despite multimodality therapy, nearly half of pts with locoregionally advanced HPV-negative HNSCC relapse. As ctDNA has the potential to identify MRD, predict outcomes, and guide treatment for these pts, we performed a retrospective cohort study to evaluate the performance of a custom-built, clinically and commercially available ctDNA assay for this population. Methods: Pts with newly diagnosed HPV-negative HNSCC (all mucosal sites) treated at a single academic institution with pre-treatment ctDNA testing (Signatera, Natera) performed during clinical care were identified. Signatera utilizes up to 16-plex PCR from matched tumor and blood to develop a personalized ctDNA assay. A subset of patients had additional ctDNA testing during follow-up. Study objectives were to understand baseline ctDNA detection in relation to disease characteristics, and to correlate ctDNA changes on- and post-treatment with disease status and survival. Results: Testing was performed in 92 of 115 (80%) pts with HPV-negative HNSCC (median age: 66, 69% male, 64% smokers); ctDNA testing could not be performed in 23 (20%) pts due to insufficient tumor tissue. Oral cavity (43%) and larynx (22%) were the most common subsites; with most having clinical T2-3 (54%) and N1-2 (51%) disease (AJCC 2017 8th edition) treated with definitive surgery (46, 40%) and/or chemoradiation (59, 51%). Pre-treatment, 69/92 (75%) pts had detectable ctDNA (range: 0.03-4049.69 mean tumor molecules/mL). No baseline independent clinical features predicted pre-treatment ctDNA detectability or levels (multiple logistic/linear regression modeling), but levels varied by T and N stage in univariate analysis (both p<0.05; Kruskal-Wallis test). At a median follow-up of 5.2 months (range: 0.2-15.1), 47 pts (51%) had >1 test result (range: 1-7; 170 total samples). Of 47 pts, 17 (36%) had ctDNA detected after treatment. Disease-free survival was significantly worse for pts who were ctDNA positive vs. negative after treatment (HR 4.35, 95%CI: 1.68-11.21, p<0.01); 1-year overall survival was 83.7% vs. 100% for pts who were ctDNA positive vs. negative. Conclusions: Tumor-informed, personalized ctDNA testing is feasible among pts with non-virally mediated HNSCC. ctDNA positivity as an early indicator of MRD positivity post-treatment was associated with inferior survival, identifying a high-risk subgroup. Further research is warranted to understand whether ctDNA may be leveraged to guide therapy and improve outcomes for HNSCC.

Abstract 3659: Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract cancer

Abstract Biliary tract cancer (BTC) is a rare type of abdominal cancer and is more frequent in Asian countries than in Western. The 5-year survival rate for BTC is very low 7 to 20% and only 20% of BTC patients could undergo curative-intent surgery. BTC diagnosis based on biopsy and cytology are low in sensitivity that prompted the necessity to identify alternative biopsy. Liquid biopsy, specifically circulating tumor DNA (ctDNA) is known for the accessibility and low invasiveness received great interest for their potential uses in various clinical applications for patients with insufficient tumor tissue, including mutation profiling, treatment monitoring, and cancer detection. However, the panel available in the market up-to-date focused mainly targeted, actionable alterations and thus not suitable for minimal residual disease (MRD) monitoring. In this study, we aimed to develop a customized ctDNA panel for MRD monitoring in BTC. In this study, 124 BTC patients undergone curative resections were enrolled at Hokkaido University and Sapporo Medical University from May 2019 to April 22. All enrolled patients have BTC diagnosis confirmed by pathological diagnosis after surgery, and without other malignant tumors. Genomic profiles of FFPE tissue were conducted using Ampliseq CCP. 13 patients were excluded for high fragmentation. Referring to the genomic profiles of 111 patients in our study and 1924 patients in TCGA established a customized panel using Ion Ampliseq HD technology. The Ampliseq HD panel claims to detect variants at 0.1% LOD for ctDNA detection. It also integrates molecular barcodes to reduce NGS-related errors. The designed panel was 258 Amplicons and 20 genes including eleven therapeutically targetable genes. Clinical characteristics of ctDNA detection were also assessed. Results:The panel validation: concordance of the mutation allele frequency (MAF) between the NGS standard control and Ampliseq HD panel showed a coefficient of determination (R2) of 0.96. Also, concordance of MAF Ampliseq CCP and Ampliseq HD panel on FFPE tumor profiling showed R2 of 0.78. When assessing 70% of BTC patients were covered to have at least one tumor-derived Ampliseq HD panel. When assessing using plasma cell-free DNA, 55% of BTC patients were detected to have at least one tumor-derived ctDNA. Distribution of detected altered top4 genes using Ampliseq HD panels shows TP53 35%, KRAS 10%, SMAD4 5%, ARID1A 2.5%. Clinical characteristics may affected the concordance of plasma and FFPE tumor tissue; stage (P=0.035), size of tumors (P=0.0088), subtype (P=0.013), LN metastasis (P=0.11). Conclusions: A highly sensitive ctDNA customized panel for BTC was established. We also found that clinical characteristics of the BTC affects the detection of ctDNA from blood liquid biopsy. Future study: MRD evaluation using this customized panel. Citation Format: Yoshihito Shinohara, Siew-Kee Low, Toru Nakamura, Yasutoshi Kimura, Takeshi Murakami, Kazuma Kiyotani, Satoshi Hirano. Development of customized circulating tumor DNA panel for minimal residual disease detection in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3659.

A Prospective, Exploratory, Phase II Study of the Xpo-1 Inhibitor Selinexor in Combination with RCHOP in Double/Triple-Hit Large B-Cell Lymphoma

Background Double-hit (DH) lymphoma or triple-hit (TH) lymphoma, according to 2016 WHO Classification Criteria, is recognized as a distinct subset of non-Hodgkin lymphoma. The optimal treatment for DH/TH lymphoma remains unclear, although outcomes are inadequate with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) alone. Selinexor is a first-in-class, oral therapeutic that forms a slowly reversible covalent bond with cytoplasm by exportin 1 (XPO1/CRM1), leading to its inactivation. The U.S. Food and Drug Administration has approved selinexor monotherapy for relapsed/refractory DLBCL. Previous studies also suggested that selinexor was used to treat 5 patients with relapsed/refractory DH lymphoma, and 1 patient achieved CR and 2 achieved PR. Here, we report on the efficacy and safety of selinexor in combination with RCHOP as first-line treatment in patients with DH or TH lymphoma. Methods This prospective, phase 2, exploratory study was conducted in Zhejiang Cancer Hospital. The primary endpoint of the study was to evaluate the objective response rate (ORR), including complete remission (CR) rate and partial remission (PR) rate. In addition, diagnostic tumor tissue was prepared to conduct NGS analysis, and dynamic peripheral blood samples were collected before treatment, after 4 cycles when achieving CR from first-line chemotherapy, and progression of disease, to conduct ctDNA analysis. The initial dose of each drug in this regimen is as follows (repeat every 21 days, 6 cycles in total): rituximab: 375mg/m 2, d0; vindesine 4mg, d1; epirubicin 75mg/m 2, d1 (or liposomal doxubicin 35mg/m 2, d1); cyclophosphamide: 750mg/m 2, d1; prednisone: 100mg, d1-5; selinexor 60mg orally once a week. AEs were assessed according to the National Cancer Institute Common Terminology Criteria (CTCAE, version 4.0). Results From May, 2022, 8 patients were enrolled in the study, including 5 females and 3 males. All patients had received at least 2 cycles of selinexor with RCHOP. A full summary of patient baseline characteristics and response to chemotherapy was shown in Table 1. Four patients have completed full cycles of chemotherapy, while 2 patients are still undergoing treatment, and 2 patients suspended subsequent chemotherapy due to rejection of antitumor treatment. A total of 34 cycles of chemotherapy have been completed. Best responses after induction as assessed by positron emission tomography/CT were 5 CRs and 3 PRs. Two patients received ASCT as consolidation therapy after achieving CR. CNS prophylaxis with intrathecal methotrexate was administered to all patients for 2-6 cycles. No patient experienced disease progression, and no patient had CNS involvement during follow-up period (Figure 1A). All patients were assessable for toxicity. All patients in the study received prophylactic granulocyte colony-stimulating factor after each cycle of chemotherapy. The most common hematologic toxicities included anemia (15/34, 44.1%), neutropenia (20/34, 58.8%), thrombocytopenia (13/34, 38.2%), leukopenia (10/34, 29.4%). Other nonhematologic toxicities included fever (6/34, 17.6%), nausea (16/34, 47.1%), vomiting (13/34, 38.2%), fatigue (20/34, 58.9%), and infection (5/34, 14.7%). NGS was conducted successfully in 3 patients, while another 5 patients could not provide NGS data due to insufficient tumor tissue. 139 somatic alterations were detected in 3 patients, with a median of 30 mutations per sample. BCL6/IGLL5/TMSB4X were the most frequently mutated gene in 100% of patients followed by 66.7% who had BTG2/ETS1/EZH2/MYC/NFKBIE/OSBPL10/SOCS1/TNFRSF14 mutations. TP53 mutations were detected in 33.3% of the patient (Figure 1B). In 71.4% (5/7) of the cases, we were able to detect a total of 143 variants in the ctDNA from pretreatment serum samples. 80 variants were detected in both tumor tissue and peripheral blood samples. 4 variants were detected only in the ctDNA, and 59 were detected only in the tissue biopsy. Serial ctDNA monitoring results showed that 100% (8/8) reduction to zero in ctDNA after 4 cycles of chemotherapy. Conclusions Selinexor combined with RCHOP may be an effective first-line regimen for patients with DH/TH lymphoma. Dynamic testing of ctDNA demonstrated deep remission to chemotherapy. Based on the encouraging results of the current study, a large scale, multicenter trial is needed to further investigate this promising regimen.

HSR23-108: Physician Preferences on Biomarker Testing in Newly-Diagnosed Metastatic NSCLC Patients Who Had Insufficient Tumor Tissue

"HSR23-108: Physician Preferences on Biomarker Testing in Newly-Diagnosed Metastatic NSCLC Patients Who Had Insufficient Tumor Tissue" published on 31 Mar 2023 by National Comprehensive Cancer Network.

Abstract P6-14-15: PD-L1 expression regulated by JAK/STAT signaling pathway contributes to cell migration and invasion in breast cancer

Abstract Background: Programmed death-ligand 1 (PD-L1) implicated in tumor immune evasion and predictive biomarker in immunotherapy is widely recognized, however, the role of PD-L1 in modulating tumor invasion remains largely unexplored. PD-L1 expression on tumor tissue is usually limited by the invasiveness, tumor heterogeneity as well as insufficient tumor tissue samples, while PD-L1 expression on circulating tumor cells (CTCs) might overcome the limitation. In the present study, we aimed to investigate the role and mechanism of PD-L1 in regulating the migration and invasion of breast cancer cells and to evaluate PD-L1 expression on CTCs in metastatic breast cancer patients. Methods: The expression level of PD-L1 in MCF-7 cells and MDA-MB-231 cells was assessed by quantitative real-time RT-PCR and Western Blot. Gain-of-function and loss-of-function study on cell migration and invasion abilities were carried out by overexpression of PD-L1 or silencing PD-L1. PD-L1 expression on CTCs in thirty-six metastatic breast cancer patients were detected. A novel staining procedure which included fluorescent glucose analog staining for CTC enumeration and immunostaining targeting CD45, vimentin and PD-L1 were analyzed. Survival curves were estimated by the Kaplan-Meier method and the log-rank test was used to compare between groups. All tests were two-sided, and p values were considered significant at the 0.05 level. Results: Compared with MCF-7 cells, PD-L1 mRNA and PD-L1 protein expression were significantly increased in MDA-MB-231 cells. Down-regulation of PD-L1 expression in MDA-MB-231 cells inhibited the migration and invasion of MDA-MB-231 cells, while overexpression of PD-L1 in MCF-7 cells increased the migration and invasion of MCF-7 cells. In addition, we found that PD-L1 expression was regulated by JAK/STAT signaling pathway. PD-L1 expression on CTCs in metastatic breast cancer patients was associated with triple negative breast cancers subtype (P=0.013). High expression of PD-L1 on CTCs in metastatic breast cancer patients was correlated to poor overall survival (HR=3.165, 95%CI: 1.121-8.938, P=0.029). Conclusion: Our results indicate that high expression of PD-L1 contributes to cell migration and invasion in breast cancer cell possibly partially through JAK/STAT signaling pathway. The PD-L1 expression on CTCs might serve as a promising non-invasive prognostic biomarker in patients with metastatic breast cancer. Citation Format: Junqing Chen. PD-L1 expression regulated by JAK/STAT signaling pathway contributes to cell migration and invasion in breast cancer [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 P6-14-15.

Preliminary Experience of Liquid Biopsy in Lung Cancer Compared to Conventional Assessment: Light and Shadows.

To assess the qualitative relationship between liquid biopsy and conventional tissue biopsy. As a secondary target, we evaluated the relationship between the liquid biopsy results and the T stage, N stage, M stage, and compared to grading. The Local Ethics Committee of the "Università degli Studi della Campania Luigi Vanvitelli", with the internal resolution number 24997/2020 of 12.11.2020, approved this spontaneous prospective study. According to the approved protocol, patients with lung cancer who underwent Fine-Needle Aspiration Cytology (FNAC), CT-guided biopsy, and liquid biopsy were enrolled. A Yates chi-square test was employed to analyze differences in percentage values of categorical variables. A p-value < 0.05 was considered statistically significant. Data analysis was performed using the Matlab Statistic Toolbox (The MathWorks, Inc., Natick, MA, USA). When a genetic mutation is present on the pathological examination, this was also detected on the liquid biopsy. ROS1 and PDL1 mutations were found in 2/29 patients, while EGFR Exon 21 was identified in a single patient. At liquid biopsy, 26 mutations were identified in the analyzed samples. The mutations with the highest prevalence rate in the study populations were: ALK (Ile1461Val), found in 28/29 patients (96.6%), EML4 (Lys398Arg), identified in 16/29 (55.2%) patients, ALK (Asp1529Glu), found in 14/29 (48.3%) patients, EGFR (Arg521Lys), found in 12/29 (41.4%) patients, ROS (Lys2228Gln), identified in 11/29 (37.9%) patients, ROS (Arg167Gln) and ROS (Ser2229Cys), identified in 10/29 (34.5%) patients, ALK (Lys1491Arg) and PIK3CA (Ile391Met), identified in 8/29 (27.6%) patients, ROS (Thr145Pro), identified in 6/29 (20.7%) patients, and ROS (Ser1109Leu), identified in 4/29 (13.8%) patients. No statistically significant differences can be observed in the mutation rate between the adenocarcinoma population and the squamous carcinoma population (p > 0.05, Yates chi-square test). We showed that, when a genetic mutation was detected in pathological examination, this was always detected by liquid biopsy, demonstrating a very high concordance rate of genomic testing between tissues and their corresponding mutations obtained by liquid biopsy, without cases of false-negative results. In addition, in our study, liquid biopsy highlighted 26 mutations, with the prevalence of ALK mutation in 96.6% of patients, supporting the idea that this approach could be an effective tool in cases with insufficient tumor tissue specimens or in cases where tissue specimens are not obtainable.

147TiP Exactis-01 - Clinical utility of returning liquid biopsy NGS results in NSCL cancer patients: A Canadian trial through the Exactis Network

Biopsy tissue analysis is central for therapeutic decision-making in the era of precision oncology. Insufficient tumor tissue and incomplete genotyping, which prevents access to targeted therapies, are considerable challenges in lung cancer patients. Liquid biopsies, a non-invasive alternative to tissue, offer more accurate capture of tumor heterogeneity and greater flexibility for real-time disease monitoring at significantly reduced cost and risk to patients. Early evidence suggests that liquid biopsies can detect actionable biomarkers in non-small cell lung cancer (NSCLC).

S131 Clinical Application of Circulating Tumor DNA in Gastrointestinal Cancer Patients

Introduction: Circulating tumor DNA (ctDNA) derived from apoptotic tumor cells is rapidly emerging as a non-invasive biomarker to detect the presence of tumor at a molecular level and guide therapy. In this study, we present our experience with this novel marker in gastrointestinal malignancy (GIM) patients. Methods: We included all patients with GIM for whom ctDNA was ordered from January 2020 to July 2021 at our center. Patient demographics, tumor type, pathology, stage, and radiographic findings were collected. Descriptive statistics were employed to report findings. Results: We identified 62 GIM patients for whom ctDNA was ordered. Results for 6 patients were not reported due to insufficient tumor tissue. Of the 56 patients for whom ctDNA results were available, 46 (82%) had colorectal adenocarcinoma (CRC) and the rest had other GI malignancies. Of the overall cohort of 56 evaluable patients, 21 (37.5%) had metastatic disease. Imaging findings paralleled ctDNA levels and correlated with tumor burden in 30 patients (53.5%). In 13 patients (23.2%), undetectable CT DNA levels influenced decisions regarding systemic therapy in addition to acting as a surrogate for treatment response. We discontinued maintenance therapy in 5 metastatic CRC patients with negative ctDNA and imaging. These patients have been off treatment for > 12 months without evidence of recurrence. We also truncated or skipped adjuvant treatment in 6 Stage III CRC patients with negative ctDNA and imaging who had significant post-operative complications or were otherwise considered high risk for severe toxicity. In one CRC patient, ctDNA was found to be undetectable post neoadjuvant therapy which corroborated with pathological complete response on surgical pathology report. 5 patients (9.09 %) had false-negative results and 1 patient had falsely elevated levels. In 6 other patients, we could not draw significant conclusions due to the availability of only a single value. Conclusion: Our experience suggests that ctDNA is not only a sensitive tool to assess tumor burden, but can also help guide therapy in locally advanced and metastatic CRC patients. However, further prospective studies are needed to improve the accuracy of test results and integrate ctDNA in day-to-day clinical practice.

Analysis of BRCA2 Copy Number Loss and Genomic Instability in Circulating Tumor Cells from Patients with Metastatic Castration-resistant Prostate Cancer

BackgroundBRCA2 alterations predict for a response to poly-ADP-ribose polymerase inhibition in metastatic castration-resistant prostate cancer (mCRPC). However, detection is hindered by insufficient tumor tissue and low sensitivity of cell-free DNA for detecting copy number loss. ObjectiveTo evaluate the BRCA2 loss detection using single-cell, shallow whole-genome sequencing (sWGS) of circulating tumor cells (CTCs) in patients with mCRPC. Design, setting, and participantsWe analyzed CTC samples collected concurrently with tumor biopsies intended for clinical sequencing in patients with progressing mCRPC. Outcome measurements and statistical analysisDifferences in proportions were evaluated using the chi-square test. Correlations between assays were analyzed in linear regression models. Associations between alterations and genomic instability were assessed on the single-cell level using mixed-effect negative binomial models. Results and limitationsWe identified 138 patients with concurrent CTC and biopsy samples. CTC sWGS generated copy number profiles in a similar proportion of patients to biopsy samples (83% vs 78%, p = 0.23), but was more effective than bone biopsies (79% vs 50%; p = 0.009). CTC sWGS detected BRCA2 loss in more patients than tissue at the ≥1 (42% vs 16%; p < 0.001) and ≥2 (27% vs 16%; p = 0.028) CTC thresholds. The overall prevalence of BRCA2 loss was not increased in CTCs using sample-level composite z scores (p = 0.4), but was significantly increased compared with a lower-than-expected prevalence in bone samples (21% vs 3%, p = 0.014). Positive/negative predictive values for CTC BRCA2 loss were 89%/96% using the ≥1 CTC threshold and 67%/92% using the composite z score. CTC BRCA2 loss was associated with higher genomic instability in univariate (1.4-fold large-scale transition difference, 95% confidence interval [CI]: 1.2–1.6; p < 0.001) and multivariable analysis (1.4-fold difference, 95% CI: 1.2–1.6; p < 0.001). ConclusionsCopy number profiles can reliably be generated using CTC sWGS, which detected a majority of tissue-confirmed BRCA2 loss and “CTC-only” losses. BRCA2 losses were supported by increases in genomic instability. Patient summaryCurrent testing strategies have limitations in their ability to detect BRCA2 loss, a relatively common alteration in prostate cancer that is used to identify patients who may benefit from targeted therapy. In this paper, we evaluated whether we could detect BRCA2 loss in individual tumor cells isolated from patient blood samples and found this method to be suitable for further analysis.

Biomarker testing in patients (pts) with metastatic colorectal cancer (mCRC): Perspectives from U.S. oncologists (ONC) in rural areas and urban clusters.

70 Background: In the US, pts living in rural areas have higher CRC mortality rates than urban areas. Clinical guidelines recommend testing for BRAF and RAS mutations and deficient mismatch repair/microsatellite instability in pts with mCRC. However, data on biomarker testing rates in rural communities compared with urban areas are limited. We surveyed ONC in the US who practice in rural areas or urban clusters to identify biomarker testing patterns and barriers (data previously reported) and conducted interviews with a select group of respondents to further understand key differences that may contribute to substandard biomarker testing rates in rural areas. Methods: A 2-part (quantitative and qualitative) survey was conducted with ONC who spend &gt; 40% of their time providing direct care to pts in rural areas or urban clusters and who had treated ≥2 pts with stage IV mCRC in the month prior to the survey. After screening, a subset of those who completed the quantitative survey participated in the qualitative survey (a 30-minute, web-assisted, telephone interview). The interview questions targeted 6 areas: clinical practice description, biomarker and genomic testing patterns, pathology and molecular tumor board, tumor tissue journey, electronic health records, and training/educational opportunities. Results: Of the 99 ONC who responded to the quantitative survey, 17 were interviewed for the qualitative survey from June 16–29, 2021. A key finding of the quantitative survey was that although ONC reported being familiar with biomarkers relevant to mCRC, the reported rate of biomarker testing was suboptimal. The interviews probed reasons why testing does not align with current guidelines and found that challenges exist throughout the tumor tissue journey including insufficient tumor tissue available for testing (especially in the relapsed/refractory setting); lack of or limited protocols, clinical decision support systems, reflexive testing, and molecular tumor boards; lengthy and difficult-to-navigate next-generation sequencing reports; and financial toxicity surrounding biomarker tests (especially for underinsured pts), among other barriers. Despite these challenges, ONC reported easy access to third-party reference labs and electronic references, such as NCCN and UpToDate. Although telehealth visits have nearly quadrupled during the COVID pandemic, access to telehealth may be limited for pts living in rural areas or urban clusters. Conclusions: The ONC surveyed reported that practicing in rural/urban clusters poses unique challenges related to tissue acquisition, practice resources, pts’ ability to pay, and clinical knowledge gaps that may affect biomarker testing rates in pts with mCRC. Addressing these gaps is warranted if optimal utilization of precision medicine tools is to be realized.

Concordancy of immunocytochemistry profiling of circulating tumor cells with immunohistochemistry for analysis of therapeutically relevant biomarkers.

3047 Background: Immunohistochemistry (IHC) profiling of tumor tissue is the present standard for evaluation of therapeutically relevant biomarkers such as ER, PR, HER2, AR, ARv7, PD-L1 and MMR for selection of targeted, endocrine and checkpoint inhibitor therapy selection. However, this critical analysis is dependent on availability of tumor tissue obtained by an invasive biopsy. Challenges to this analysis include insufficient tumor tissue and inability to perform a repeat biopsy to obtain fresh tumor tissue. We have previously described an approach for negative enrichment of Circulating Tumor Cells (CTCs) from peripheral blood and for Immunocytochemistry (ICC) profiling of these CTCs for detection of diagnostically relevant tissue of origin and subtype specific markers, concordant with tumor tissue analysis. Methods: In the present study, we determined concordance between tumor tissue (HPE) and CTCs (ICC) for ascertaining the status of therapeutically relevant markers ER, PR, HER2, AR, ARv7 PD-L1 and MMR. We evaluated 201 matched pairs of tumor tissue (FFPE blocks) and CTCs obtained from peripheral blood. Results: Among the 743 paired assays on matched tumor tissue and CTCs, concordance (positive or negative) was observed in 651 matched pairs (87.6%). The concordance was 82.9% for ER, 100% for PR, 90.2 % for Her2, 93.8% for AR, 90% for Arv7, 85.1% and 87.6% for PD-L1 clones 22c3 and 28-8, and 85.6% for MMR (MLH1, MSH2, MSH6, and PMS2). Conclusions: The study findings indicate that ICC analysis of CTCs may be able to substitute IHC analysis of tumor tissue for profiling of therapeutically relevant markers. This approach may have application in cases where tumor tissue may be limiting and / or where an invasive biopsy to obtain tumor tissue may be unviable.

Accuracy of endoscopic ultrasound-guided needle aspiration specimens for molecular diagnosis of non-small-cell lung carcinoma.

BACKGROUNDEndoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are highly sensitive for diagnosing and staging lung cancer. In recent years, targeted therapy has shown great significance in the treatment of non-small cell lung carcinoma (NSCLC). Using these minimally invasive techniques to obtain specimens for molecular testing will provide patients with a more convenient diagnostic approach.AIMTo evaluate the feasibility and accuracy of tissue samples obtained using EUS-FNA and EBUS-TBNA for molecular diagnosis of NSCLC.METHODSA total of 83 patients with NSCLC underwent molecular testing using tissues obtained from EUS-FNA or EBUS-TBNA at the Tianjin Medical University Cancer Hospital from January 2017 to June 2019. All enrolled patients underwent chest computed tomography or positron emission tomography/computed tomography prior to puncture. We detected abnormal expression of EGFR, KRAS, MET, HER2, ROS1 and anaplastic lymphoma kinase protein. Two patients failed to complete molecular testing due to insufficient tumor tissue. The clinical features, puncture records, molecular testing results and targeted treatment in the remaining 81 patients were summarized.RESULTSIn a total of 99 tissue samples obtained from 83 patients, molecular testing was successfully completed in 93 samples with a sample adequacy ratio of 93.9% (93/99). Biopsy samples from two patients failed to provide test results due to insufficient tumor tissue. In the remaining 81 patients, 62 cases (76.5%) were found to have adenocarcinoma, 11 cases (13.6%) had squamous cell carcinoma, 3 cases (3.7%) had adenosquamous carcinoma and 5 cases (6.2%) had NSCLC-not otherwise specified. The results of molecular testing showed EGFR mutations in 21 cases (25.9%), KRAS mutations in 9 cases (11.1%), ROS-1 rearrangement in 1 case (1.2%) and anaplastic lymphoma kinase-positive in 5 cases (6.2%). Twenty-four patients with positive results received targeted therapy. The total effectiveness rate of targeted therapy was 66.7% (16/24), and the disease control rate was 83.3% (20/24).CONCLUSIONTissue samples obtained by EUS-FNA or EBUS-TBNA are feasible for the molecular diagnosis of NSCLC and can provide reliable evidence for clinical diagnosis and treatment.

Assessment of PD-L1 expression in circulating tumor cells in patients with breast cancer.

e13001 Background: Evidence is accumulating that the immune checkpoint blockade targeting the programmed death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis has improved progression-free and overall survival of advanced breast cancer patients. PD-L1 tumor expression is emerging as a potential biomarker in PD-1/PD-L1 checkpoint blockade therapies. However, PD-L1 expression on tumor tissue is limited by the temporal and spatial heterogeneity as well as insufficient tumor tissue samples. PD-L1 expression in circulating tumor cells (CTCs) might overcome the limitation. We investigated the utility of CTCs as a noninvasive method to evaluate PD-L1 status in breast cancer patients. Methods: 46 breast cancer patients including 42 metastatic breast cancer patients and four early breast cancer patients were enrolled. CTCs were collected from peripheral blood samples using a microfluidic-based CTC separator. A novel staining procedure which included fluorescent glucose analog staining and immunostaining targeting CD45, vimentin and PD-L1 were analyzed. PD-L1 expression level was analyzed as the fraction of PD-L1 positive CTCs among total CTCs. Results: 46 breast cancer patients all had detected CTCs, ranging from 12 to 110. The fraction of PD-L1 positive CTCs varied from 2.08% to 85.69%. PD-L1 expression was obviously higher in triple negative breast cancer (TNBC) patients as compared with non-TNBC patients (47.16% vs 26.23%, P &lt; 0.01). Interestingly, we observed that PD-L1 positive CTCs were also detected in four early TNBC patients. No significant association was found between PD-L1 expression and mesenchymal CTC phenotype. Conclusions: These data suggest that assessment of PD-L1 expression in CTCs from breast cancer patients is feasible. PD-L1 expression in CTCs might serve as a complementary tool for PD-L1 expression analysis in breast cancer patients.

Lessons learned from clinical trial queries on small biopsy collections: importance of rapid on-site evaluation

Small biopsies and cytology specimens have become increasingly important for clinical trials and biomarker testing. Thus, institutions must ensure that adequate lesional material meeting the specifications for a multitude of different protocols is available. This can be achieved using rapid on-site evaluation (ROSE). The aim of the present study was to determine the recent clinical trial biopsy characteristics and study the feedback on these collections at our institution. Clinical trial biopsies performed at our institution and trial feedback (including "queries") were analyzed from the 2017 to 2019. The query data were reviewed in detail, in addition to any protocol modifications related to biopsy requirements and study protocol changes. A total of 698 biopsy collections were performed for clinical trial purposes for 95 trials, with most requiring biopsies at >1 time point (63.2%), for phase I or II trials (92.6%), and for specific tumor types (67.4%). Only 18 of the trials (18.9%) requiring fresh tissue biopsies provided feedback. The feedback included data from 90 cases (12.9%), of which 27 (30.0%) had queries regarding insufficient (n = 10; 37.0%) or borderline (n = 17; 63.0%) tumor tissue. Only 1 (3.7%) of these had had ROSE by cytology. ROSE was performed in accordance with institutional guidelines (45.3%), as required by the study (1.1%), or because of trial modification (5.3%). The present investigation has shown the high volume of clinical trial biopsies managed at our academic cancer center. Feedback from the trials was low at 18.9% and frequently involved suboptimal cases without ROSE used at acquisition. This has led to more widespread adoption of ROSE to mitigate insufficient biopsy specimens and repeat procedures. The high volume of clinical trial biopsies and variability in trial needs necessitates a collaborative multidisciplinary network, including cytology services, to facilitate these important biopsies for patients with cancer.

Prospective assessment of the clinical benefit of a tailored cancer gene set built on a next-generation sequencing platform in patients with recurrent or metastatic head and neck cancer.

We performed a prospective trial to assess the clinical benefit of a tailored gene set built on a next-generation sequencing (NGS) platform in patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Archived tumor tissue obtained from patients with recurrent or metastatic HNSCC was analyzed for variants by a tailored Comprehensive Cancer Gene set of 40 genes (CCG-40) performed on a NGS platform. These data were provided to clinicians to inform treatment decisions. The primary endpoint was clinical benefit (disease control) that resulted from selection and administration of a targeted therapy based on results of the CCG-40. Barriers to performance and implementation of the assay were recorded. Forty patients enrolled. Primary tumor sites included oropharynx (14), larynx/hypopharynx (14), oral cavity (9), and nasopharynx (3). The CCG-40 assay was performed in 23 patients (57.5%), but not in 17 patients due inadequate financial coverage (12) or insufficient tumor tissue (5). Potentially actionable tumor variants were identified in 3 patients (7.5%); all were PIK3CA variants. Due to inability to obtain access to candidate drugs (2) or rapid decline in performance status (1), none of these patients received targeted therapy informed by the CCG-40 results. The CCG-40 assay did not provide clinical benefit to the patients on this trial. Identification of limitations of the assay and barriers to the test's performance and application may be used to optimize this strategy in future trials.

P1.09-06 Evaluation of Molecular Testing in a Dutch Cohort of Metastatic Non-Small Cell Lung Cancer Patients from 2017

Adequate and timely testing for genetic alterations in non-small cell lung cancer (NSCLC) is necessary to consider targeted therapy when a certain genetic alteration is present. Previously, we demonstrated that in the Netherlands molecular testing was suboptimal in 2015, as 25% (EGFR/KRAS and ALK) to 50% (ROS1) of patients were not tested according to the guidelines, and notable variation between laboratories was present. Currently, by analyzing a cohort of metastatic NSCLC from 2017 we aim to assess whether the performance of molecular testing improved. All fully registered stage IV non-squamous NSCLC with incidence year 2017 from the Netherlands Cancer Registry were matched to the Dutch pathology registry (PALGA). Using information extracted from pathology excerpts, proportions of tumors tested for EGFR and/or KRAS, BRAF, and HER2 mutation, and ALK, ROS1, and RET rearrangement within 3 months after diagnosis were determined, and reason for not testing were assessed. Of 2596 identified patients, we have currently analyzed 511 (20%). Twenty-three patients were non-eligible after matching, leaving 488 patients. Of these patients, 262 (54%) were male and 413 (88%) had an adenocarcinoma. EGFR and/or KRAS testing was performed within 3 months after diagnosis in 412 patients (84.4%). Of the EGFR/KRAS wildtype tumors (n=184), 167 (90.8%) were tested for BRAF, 158 (85.9%) for HER2, 157 (85.3%) for ALK, 110 (59.8%) for ROS1, and 73 (39.7%) for RET. Insufficient tumor tissue and inappropriate specimen were the most stated reasons for not testing. These preliminary data show significantly higher testing proportions for EGFR and/or KRAS and ALK as compared to 2015. Further improvement remains possible to identify candidates for targeted therapy. At the WCLC meeting, we expect to present the variation between laboratories for the entire cohort.

Circulating Tumor Cell PD-L1 Expression as Biomarker for Therapeutic Efficacy of Immune Checkpoint Inhibition in NSCLC.

Over the last decade, the immune checkpoint blockade targeting the programmed death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis has improved progression-free and overall survival of advanced non-small cell lung cancer (NSCLC) patients. PD-L1 tumor expression, along with tumor mutational burden, is currently being explored as a predictive biomarker for responses to immune checkpoint inhibitors (ICIs). However, lung cancer patients may have insufficient tumor tissue samples and the high bleeding risk often prevents additional biopsies and, as a consequence, immunohistological evaluation of PD-L1 expression. In addition, PD-L1 shows a dynamic expression profile and can be influenced by intratumoral heterogeneity as well as the immune cell infiltrate in the tumor and its microenvironment, influencing the response rate to PD-1/PD-L1 axis ICIs. Therefore, to identify subgroups of patients with advanced NSCLC that will most likely benefit from ICI therapies, molecular characterization of PD-L1 expression in circulating tumor cells (CTCs) might be supportive. In this review, we highlight the use of CTCs as a complementary diagnostic tool for PD-L1 expression analysis in advanced NSCLC patients. In addition, we examine technical issues of PD-L1 measurement in tissue as well as in CTCs.

Liquid biopsy assay for lung carcinoma using centrifuged supernatants from fine-needle aspiration specimens.

Tumor mutation profiling is standard-of-care in lung carcinoma patients. However, comprehensive molecular profiling of small specimens, including core needle biopsy (CNB) and fine-needle aspiration (FNA) specimens, may often be inadequate due to limited tissue. Centrifuged FNA supernatants, which are typically discarded, have emerged recently as a novel liquid-based biopsy for molecular testing. In this study, we evaluate the use of lung carcinoma FNA supernatants for detecting clinically relevant mutations. Supernatants from lung carcinoma FNA samples (n = 150) were evaluated. Samples were further analyzed using next-generation sequencing (NGS) and ultrasensitive droplet digital PCR (ddPCR). Mutation profiles in a subset of samples were compared with results derived from paired tissue samples from the same patient (n = 67) and available plasma liquid biopsy assay (n = 45). All 150 samples yielded adequate DNA and NGS were carried out successfully on 104 (90%) of 116 selected samples. Somatic mutations were detected in 82% of the samples and in 50% of these patients a clinically relevant mutation was identified that would qualify them for targeted therapy or a clinical trial. There was high overall concordance between the mutation profiles of supernatants and the corresponding tissue samples, with 100% concordance with concurrent FNA and 96% with concurrent CNB samples. Comparison of actionable driver mutations detected in supernatant versus plasma samples showed 84% concordance. FNA supernatants can provide a valuable specimen source for genotyping lung carcinoma especially in patients with insufficient tumor tissue, thereby reducing multigene mutation profiling failure rates, improving turnaround times, and avoiding repeat biopsies.