Multigene Assays Research Articles

An update on the pathological classification of breast cancer.

Breast cancer (BC) is a heterogeneous disease, encompassing a diverse spectrum of tumours with varying morphological, biological, and clinical phenotypes. Although tumours may show phenotypic overlap, they often display different biological behaviour and response to therapy. Advances in high-throughput molecular techniques and bioinformatics have contributed to improved understanding of BC biology and refinement of molecular taxonomy with the identification of specific molecular subclasses. Although the traditional pathological morphological classification of BC is of paramount importance and provides diagnostic and prognostic information, current interest focusses on the use of a single gene and multigene assays to stratify BC into distinct groups to guide decisions on systemic therapy. This review considers approaches to the classification of BC, including their limitations, and with particular emphasis on the fundamental role of morphology in establishing an accurate diagnosis of primary invasive carcinoma of breast origin. This forms the basis for further morphological characterization and for all other approaches to BC classification that are used to provide prognostic and therapeutic predictive information.

Association of Body Mass Index With 21-Gene Recurrence Score Among Women With Estrogen Receptor–Positive, ERBB2–Negative Breast Cancer

Body mass index (BMI) may affect the 21-gene recurrence score (RS) in patients with ER-positive, ERBB2-negative breast cancer. If high BMI increases genomic risk in ER-positive, ERBB2-negative breast cancer, weight control will become more important. To assess the association between RS and BMI according to age groups and address BMI as a factor associated with high RS. This cohort study included 2295 patients with ER-positive, ERBB2-negative breast cancer who had undergone a multigene assay between March 29, 2010, and December 31, 2020, in 2 hospitals. All of the study patients were Korean women, and the median follow-up period was 45 months (range, 1-40 months). The correlations between continuous RS and BMI were investigated. A high BMI was defined as a body mass index greater than or equal to 25. In the younger age group (age ≤45 years), a high RS was defined as an RS of greater than 20. Body mass index. The Pearson correlation coefficient was used to estimate the association between RS and BMI. A multivariable binary logistic model was used to identify high RS. Among the 2295 women included (mean [SD] age, 49.8 [4.00] years; range, 22-81 years), 776 were aged 45 years or younger; RS and BMI were weakly correlated (correlation coefficient, 0.119; P < .001) in this younger group. Among them, the proportion of patients with an RS greater than 20 was significantly higher in the high BMI group than in the normal BMI group (45.5% [46 of 101] vs 27.3% [184 of 675]; P < .001). In the multivariable analysis, high BMI was an associated factor for high RS (odds ratio, 2.06; 95% CI, 1.28-3.32; P = .003). The 21-gene multigene assay-guided chemotherapy rate was significantly higher in patients with high BMI (30.7% [31 of 101] vs 20.2% [136 of 674]; P = .02). In this cohort study of women aged 45 years or younger, high BMI was associated with higher RS in those with ER-positive, ERBB2-negative breast cancer; further studies are necessary to examine the underlying mechanisms.

Diagnostic yield of next generation sequencing gene panel assays for early-onset glaucoma in an ethnically diverse population

Early-onset glaucoma is a potentially sight-threatening condition with high heritability. Next generation sequencing is a cost-effective alternative to individual gene screening that could expedite its diagnosis. However, the diagnostic yield of multigene panel assays for early-onset glaucoma varies according to the tested population. The purpose of this study was to ascertain the diagnostic yield of next generation sequencing panels in our cohort and to identify population characteristics that increase such yield. We conducted a retrospective review of the medical records of consecutive patients from November 2016 to August 2021 who were evaluated at our clinics for early-onset glaucoma and had undergone next generation sequencing panels for molecular diagnosis. A total of 118 patients were included, in 22 of whom (19%) a causative variant was identified. Diagnostic yield varied by age of onset: of 60 patients with onset at <3 years of age, 19 (32%) had such variants identified. In contrast, of 58 patients with later-onset glaucoma, 3 (5%) had said variants identified (P = 0.0003). Other metrics that increased diagnostic yield were presence of additional ocular anomalies (P = 0.0092) and identifying ethnicity as White (compared with non-White, P = 0.0001). In childhood glaucoma, earlier age of onset is correlated with higher likelihood of pathogenic variant identification. The large proportion of unsolved cases indicates a robust opportunity for gene discovery and genetic therapy targets in early-onset glaucoma patients.

273 (PB-097) Poster - Real-world use of multigene signatures in early breast cancer: the experience by the Lombardy Genomic Assays for Breast Cancer Working Group

Background: Multigene assays (MGAs) are extremely useful tools for tailoring adjuvant chemotherapy (CT) in ER+/HER2- EBC. In July 2019, Lombardy was the first Region in Italy to reimburse MGAs for patients (pts) with formal indication to receive adjuvant CT after multidisciplinary discussion. Here, we report real-world MGAs experience in 6 referral Cancer Centers in Lombardy.

Recurrence Score® Result Impacts Treatment Decisions in Hormone Receptor-Positive, HER2-Negative Patients with Early Breast Cancer in a Real-World Setting-Results of the IRMA Trial.

Patients with hormone receptor-positive (HR+), HER2-negative (HER2-) early breast cancer (eBC) with a high risk of relapse often undergo adjuvant chemotherapy. However, only a few patients will gain benefit from chemotherapy. Since classical tumor characteristics (grade, tumor size, lymph node involvement, and Ki67) are of limited value to predict chemotherapy efficacy, multigene expression assays such as the Oncotype DX® test were developed to reduce over- and undertreatment. The IRMA trial analyzed the impact of Recurrence Score® (RS) assessment on adjuvant treatment recommendations. The RS result was assessed in patients with HR+/HER2- unilateral eBC with 0-3 pathologic lymph nodes who underwent primary surgical treatment at the Department for Women's Health of Tuebingen University, Germany. Therapy recommendations without knowledge of the RS result were compared to therapy recommendations with awareness of the RS result. In total, 245 patients underwent RS assessment. Without knowledge of the RS result, 92/245 patients (37.6%) would have been advised to receive chemotherapy. After RS assessment, 56/245 patients (22.9%) were advised to undergo chemotherapy. Chemotherapy was waived in 47/92 patients (51.1%) that were initially recommended to receive it. Chemotherapy was added in 11/153 patients (7.2%) that were recommended to not receive it initially. Using the RS result to guide adjuvant treatment decisions in HR+/HER2- breast cancer led to a substantial reduction of chemotherapy. In view of the results achieved in prospective studies, the RS result is among other risk-factors suitable for the individualization of adjuvant systemic therapy.

Clinical utility of liquid biopsy-based companion diagnostics in the non-small-cell lung cancer treatment

Recently, technological advances in the detection and biological characterization of circulating tumor DNA (ctDNA) have enabled the implementation of liquid biopsy testing into clinical practice. Methods for analysis of liquid biopsies have rapidly evolved over the past few years and have continued to advance, thus providing details about tumor biological characteristics such as tumor progression, metastasis, tumor heterogeneity, genomic mutation profile, clonal evolution, etc. In tandem with technological advances, the implementation of liquid biopsy in routine clinical settings has proceeded. In 2016, the Food and Drug Administration (FDA) approved the first ctDNA liquid biopsy test to detect epidermal growth factor receptor (EGFR) gene mutations in patients with non-small-cell lung cancer (NSCLC) as a companion diagnostic for molecular targeted drug of EGFR-tyrosine kinase inhibitor (TKI, EGFR-TKI). More recently, multigene panel assays of liquid biopsy have been approved as companion diagnostics and have been used in routine clinical settings. The estimation of blood tumor mutation burden (bTMB) to predict the efficacy of immune checkpoint inhibitor (ICI) treatment can be one of the promising approaches to liquid biopsy. The next stage of implementation of liquid biopsy for routine clinical settings is for monitoring of ctDNA after surgical treatment to predict prognosis and to detect disease relapse earlier than conventional imaging diagnosis. Its clinical utility is under assessment in several clinical trials. This review introduces recent advances in liquid biopsy methodology, the development of biomarkers, and its clinical utility in the treatment of NSCLC patients.

Intrinsic subtypes in Ethiopian breast cancer patient

PurposeThe recent development of multi-gene assays for gene expression profiling has contributed significantly to the understanding of the clinically and biologically heterogeneous breast cancer (BC) disease. PAM50 is one of these assays used to stratify BC patients and individualize treatment. The present study was conducted to characterize PAM50-based intrinsic subtypes among Ethiopian BC patients.Patients and methodsFormalin-fixed paraffin-embedded tissues were collected from 334 BC patients who attended five different Ethiopian health facilities. All samples were assessed using the PAM50 algorithm for intrinsic subtyping.ResultsThe tumor samples were classified into PAM50 intrinsic subtypes as follows: 104 samples (31.1%) were luminal A, 91 samples (27.2%) were luminal B, 62 samples (18.6%) were HER2-enriched and 77 samples (23.1%) were basal-like. The intrinsic subtypes were found to be associated with clinical and histopathological parameters such as steroid hormone receptor status, HER2 status, Ki-67 proliferation index and tumor differentiation, but not with age, tumor size or histological type. An immunohistochemistry-based classification of tumors (IHC groups) was found to correlate with intrinsic subtypes.ConclusionThe distribution of the intrinsic subtypes confirms previous immunohistochemistry-based studies from Ethiopia showing potentially endocrine-sensitive tumors in more than half of the patients. Health workers in primary or secondary level health care facilities can be trained to offer endocrine therapy to improve breast cancer care. Additionally, the findings indicate that PAM50-based classification offers a robust method for the molecular classification of tumors in the Ethiopian context.

A novel germline mutation of TP53 with breast cancer diagnosed as Li–Fraumeni syndrome

TP53 is a tumor suppressor gene and, when dysfunctional, it is known to be involved in the development of cancers. Li–Fraumeni syndrome (LFS) is a hereditary tumor with autosomal dominant inheritance that develops in people with germline pathogenic variants of TP53. LFS frequently develops in parallel to tumors, including breast cancer. We describe a novel germline mutation in TP53 identified by performing a multi-gene panel assay in a breast cancer patient with bilateral breast cancer.

Molecular characterization of renal cell carcinoma tumors from a phase III anti-angiogenic adjuvant therapy trial

Multigene assays can provide insight into key biological processes and prognostic information to guide development and selection of adjuvant cancer therapy. We report a comprehensive genomic and transcriptomic analysis of tumor samples from 171 patients at high risk for recurrent renal cell carcinoma post nephrectomy from the S-TRAC trial (NCT00375674). We identify gene expression signatures, including STRAC11 (derived from the sunitinib-treated population). The overlap in key elements captured in these gene expression signatures, which include genes representative of the tumor stroma microenvironment, regulatory T cell, and myeloid cells, suggests they are likely to be both prognostic and predictive of the anti-angiogenic effect in the adjuvant setting. These signatures also point to the identification of potential therapeutic targets for development in adjuvant renal cell carcinoma, such as MERTK and TDO2. Finally, our findings suggest that while anti-angiogenic adjuvant therapy might be important, it may not be sufficient to prevent recurrence and that other factors such as immune response and tumor environment may be of greater importance.

The utility of a genetic progression risk test for Barrett esophagus.

This study sought to characterize the utility of a gene methylation-based biomarker test that has been validated to predict progression towards esophageal adenocarcinoma. Barrett esophagus (BE) is a precursor condition for esophageal adenocarcinoma (EAC) with somewhat variable approaches among gastroenterologists toward managing neoplastic progression risk. Capsulomics has developed a validated multigene DNA methylation-based biomarker assay performed on BE biopsies designed to address this variability by classifying BE patients into progression risk groups. In the current study, a survey was administered to practicing gastroenterologists in order to assess the potential impact of this assay on clinical practice. In this context, 89% (95% Cl: 85.4-92.6%) of surveyed physicians felt strongly that the multigene Barrett Esophagus test helped resolve uncertainties and optimize care of patients with BE by impacting their decisions on surveillance intervals and use of active treatments, such as ablation. The assay significantly impacted surveillance intervals for both high-risk (22.0 no assay vs 12.3 months with assay; P = 1.7E-8) and low-risk (7.9 no assay vs 11.4 months with assay, P = 8.8E-4) stratified case results. Finally, the assay also significantly impacted decisions to pursue active ablation treatments in both high-risk (5% recommending ablation without assay vs 42% with assay; P = 3.7E-11) and low-risk (42% recommending ablation without assay vs 29% with assay; P = .049) stratified case results. Results demonstrated a strong effect of the assay on clinical decision making, even in conjunction with established clinical guidelines.

150P Assessment of high Ki67 (≥20%) ER+HER2- breast cancer with a 21-gene multigene assay

Ki67 immunohistochemistry (IHC) with a cut-off of 20% by MIB-1 PharmDx assay has been approved as a companion diagnostic for adjuvant abemaciclib in high-risk ER+HER2- breast cancer. We addressed the genomic risk profile of high Ki67 (≥20%) ER+HER2- breast cancer using a 21-gene multigene assay (Oncotype DX test). In addition, we investigated survival outcomes and identified factors associated with low genomic risk in the high Ki67 group.

Cost-Effectiveness Analysis of the Oncotype DX Breast Recurrence Score® Test in Node-Negative Early Breast Cancer.

BackgroundThe 21-gene assay (the Oncotype DX Breast Recurrence Score® test) is a validated multigene assay which produces the Recurrence Score® result (RS) to inform decisions on the use of adjuvant chemotherapy in human epidermal growth factor receptor 2-negative (HER2-), hormone receptor positive (HR+) early invasive breast cancer. A model-based economic evaluation estimated the cost-effectiveness of the 21-gene assay against the use of clinical risk tools alone based on the latest evidence from prospective studies.MethodsThe proportion of patients assigned to chemotherapy conditional on their RS result was obtained from retrospective data from the Clalit registry. The probability of distant recurrence with endocrine and chemo-endocrine therapy conditional on RS result was obtained from TAILORx and NSABP B-20 trials. The cost-effectiveness of the 21-gene assay compared to using clinical risk tools alone was estimated in terms of cost per quality-adjusted life-year (QALY) over a lifetime horizon.ResultsThe 21-gene assay was more effective (0.17 more quality-adjusted life years) at a lower cost (-£519) over a lifetime compared to clinical risk alone. The model results were sensitive to assumptions around the magnitude of benefit of chemotherapy in the high RS result subgroup. Other assumptions underpinning the model, such as the proportion of patients assigned to chemotherapy in the low and mid-range RS result subgroups and long-term distant recurrence probabilities, had a smaller impact on the results.ConclusionThe analysis showed that the cost-effectiveness of the 21-gene assay is sensitive to assumptions for chemotherapy sparing for patients with RS 0–25 whose outcomes with endocrine therapy are no worse compared to chemotherapy-assigned patients, and a chemotherapy benefit in the RS 26–100 group. Future studies need to incorporate a wider set of tumour profiling tests other than the 21-gene assay to allow a direct comparison of their cost-effectiveness.

Diagnostic and Therapeutic Potential of Circulating-Free DNA and Cell-Free RNA in Cancer Management.

Over time, molecular biology and genomics techniques have been developed to speed up the early diagnosis and clinical management of cancer. These therapies are often most effective when administered to the subset of malignancies harboring the target identified by molecular testing. Important advances in applying molecular testing involve circulating-free DNA (cfDNA)- and cell-free RNA (cfRNA)-based liquid biopsies for the diagnosis, prognosis, prediction, and treatment of cancer. Both cfDNA and cfRNA are sensitive and specific biomarkers for cancer detection, which have been clinically proven through multiple randomized and prospective trials. These help in cancer management based on the noninvasive evaluation of size, quantity, and point mutations, as well as copy number alterations at the tumor site. Moreover, personalized detection of ctDNA helps in adjuvant therapeutics and predicts the chances of recurrence of cancer and resistance to cancer therapy. Despite the controversial diagnostic values of cfDNA and cfRNA, many clinical trials have been completed, and the Food and Drug Administration has approved many multigene assays to detect genetic alterations in the cfDNA of cancer patients. In this review, we underpin the recent advances in the physiological roles of cfDNA and cfRNA, as well as their roles in cancer detection by highlighting recent clinical trials and their roles as prognostic and predictive markers in cancer management.

RNA sequencing-based single sample predictors of molecular subtype and risk of recurrence for clinical assessment of early-stage breast cancer

Multigene assays for molecular subtypes and biomarkers can aid management of early invasive breast cancer. Using RNA-sequencing we aimed to develop single-sample predictor (SSP) models for clinical markers, subtypes, and risk of recurrence (ROR). A cohort of 7743 patients was divided into training and test set. We trained SSPs for subtypes and ROR assigned by nearest-centroid (NC) methods and SSPs for biomarkers from histopathology. Classifications were compared with Prosigna in two external cohorts (ABiM, n = 100 and OSLO2-EMIT0, n = 103). Prognostic value was assessed using distant recurrence-free interval. Agreement between SSP and NC for PAM50 (five subtypes) was high (85%, Kappa = 0.78) for Subtype (four subtypes) very high (90%, Kappa = 0.84) and for ROR risk category high (84%, Kappa = 0.75, weighted Kappa = 0.90). Prognostic value was assessed as equivalent and clinically relevant. Agreement with histopathology was very high or high for receptor status, while moderate for Ki67 status and poor for Nottingham histological grade. SSP and Prosigna concordance was high for subtype (OSLO-EMIT0 83%, Kappa = 0.73 and ABiM 80%, Kappa = 0.72) and moderate and high for ROR risk category (68 and 84%, Kappa = 0.50 and 0.70, weighted Kappa = 0.70 and 0.78). Pooled concordance for emulated treatment recommendation dichotomized for chemotherapy was high (85%, Kappa = 0.66). Retrospective evaluation suggested that SSP application could change chemotherapy recommendations for up to 17% of postmenopausal ER+/HER2-/N0 patients with balanced escalation and de-escalation. Results suggest that NC and SSP models are interchangeable on a group-level and nearly so on a patient level and that SSP models can be derived to closely match clinical tests.

Circulating Tumor DNA-Based Genomic Profiling Assays in Adult Solid Tumors for Precision Oncology: Recent Advancements and Future Challenges.

Simple SummaryThe use of liquid biopsy for tumor genomic profiling to identify genomic biomarkers for targeted therapies has revolutionized the clinical practice in oncology management. In this review, we have summarized the recent advancements of liquid biopsy-based genomic profiling that have led to their approval for treatment selection in advanced cancer patients and highlighted the major factors that should be considered to choose the most appropriate genomic profiling assay for different patients under different clinical conditions.Genomic profiling using tumor biopsies remains the standard approach for the selection of approved molecular targeted therapies. However, this is often limited by its invasiveness, feasibility, and poor sample quality. Liquid biopsies provide a less invasive approach while capturing a contemporaneous and comprehensive tumor genomic profile. Recent advancements in the detection of circulating tumor DNA (ctDNA) from plasma samples at satisfactory sensitivity, specificity, and detection concordance to tumor tissues have facilitated the approval of ctDNA-based genomic profiling to be integrated into regular clinical practice. The recent approval of both single-gene and multigene assays to detect genetic biomarkers from plasma cell-free DNA (cfDNA) as companion diagnostic tools for molecular targeted therapies has transformed the therapeutic decision-making procedure for advanced solid tumors. Despite the increasing use of cfDNA-based molecular profiling, there is an ongoing debate about a ‘plasma first’ or ‘tissue first’ approach toward genomic testing for advanced solid malignancies. Both approaches present possible advantages and disadvantages, and these factors should be carefully considered to personalize and select the most appropriate genomic assay. This review focuses on the recent advancements of cfDNA-based genomic profiling assays in advanced solid tumors while highlighting the major challenges that should be tackled to formulate evidence-based guidelines in recommending the ‘right assay for the right patient at the right time’.

The Senologic International Society Survey on Ductal Carcinoma In Situ: Present and Future.

Therapeutic management of ductal carcinoma in situ (DCIS) is heterogeneous among countries worldwide, and some treatment indications are still controversial. To investigate DCIS management in different countries; identify both consensual practices and controversial topics; and survey opinions about the future management of DCIS. The Senologic International Society network members participated to an online survey using a questionnaire, between November 2021 and February 2022. Twenty-two responses from 20 different countries showed that organized breast cancer screening programs were present for 87% participants, and DCIS cases represented 13.7% of all breast cancers. Most participants used the grade classification (100%), the morphological classification (78%) and performed immunohistochemistry assays (73%). In case of conservative treatment, the mean re-excision rate was 10.3% and clear margins of mean 2.5 mm were considered healthy. Radical mastectomy rate was 35.5% with a breast reconstruction rate of 53%. Tumor bed boost indications were heterogeneous, and 73% of participants indicated hormone therapy for hormone-positive DCIS. Surgery and radiotherapy omission for some low-risk DCIS were considered by 73% of participants. Multigene assays were used by 43% of participants. Concerning future changes in DCIS management, participants mostly answered surgical de-escalation (48%), radiotherapy de-escalation (35) and/or active surveillance for some cases (22%). This survey provided an overview of the current practices of DCIS management worldwide. It showed that some areas are rather consensual: incidence increases over time, treatment in young women, pathological classifications, definition of healthy margins, the skin-sparing mastectomy and immediate breast reconstruction. However, some topics are still debated and result in heterogeneous practices, such as evolution in the age of diagnosis, the benefit of de-escalation in low-risk DCIS among elderly women, indications for hormone therapy, radiotherapy omission, or multigene assays. Further evidence is needed to reach consensus on these points, and innovative approaches are still under evaluation in clinical trials. The International Senologic Society, by its members, encourages precision medicine and personalized treatments for DCIS, to avoid overtreatment and overdiagnosis, and provide better healthcare to women with DCIS.

SY19-4 Precision oncology for adjuvant treatment of colon cancer

Accurate prediction of recurrence risk is extremely important for tailoring adjuvant chemotherapy for individual colon cancer patients. To date, some multi-gene assay systems have been developed to evaluate recurrence risk in patients with colon cancer. The 12-gene Recurrence Score assay (Oncotype DX) for colon cancer has been validated in stage II and III colon cancer as well as breast cancer. Another micro array based assay system, 55-gene classifier (55GC) for colon cancer has also been validated in resected stage II and stage III treated with adjuvant chemotherapy in Japan.

EE92 Cost-Effectiveness Analysis of the Oncotype DX Breast Recurrence Score® Test for HR+/HER2- Early-Stage Breast Cancer in the US

The Oncotype DX Breast Recurrence Score® (RS) test is a multigene assay providing information to personalize treatment plans for patients with HR+/HER2- early-stage breast cancer. As demonstrated in the TAILORx and RxPONDER prospective randomized clinical trials, the Oncotype DX test can identify patients who are unlikely to benefit from adjuvant chemotherapy. The cost-effectiveness of decisions based on the Oncotype DX test compared to clinic-pathologic risk assessment alone was assessed from a US Medicare perspective.

Clinical Outcomes of Hypofractionated Whole Breast Irradiation in Early Stage, Biologically High-Risk Breast Cancer

PurposeAdoption of hypofractionated whole breast irradiation (HWBI) for patients with early-stage, biologically high-risk breast cancer remains relatively low. We compared clinical outcomes of conventionally fractionated whole breast irradiation (CWBI) versus moderate HWBI in this patient population. Methods and MaterialsWe queried a prospectively maintained database for patients with early-stage (T1-2, N0, M0) breast cancer who received whole breast irradiation with either CWBI or moderate HWBI at a single institution. We included only patients with biologically high-risk tumors (defined as either estrogen receptor/progesterone receptor/human epidermal growth factor receptor 2 negative, human epidermal growth factor receptor 2 amplified, and/or patients with a high-risk multigene assay) who received systemic chemotherapy. Inverse probability of treatment weighting was used to compare treatment cohorts and to estimate 5-year time to event endpoints. Hazard ratios (HR) and 95% confidence interval (CI) were determined based on Cox proportional hazards model. ResultsWe identified 300 patients, of whom 171 received CWBI and 129 received HWBI. There was a statistically significant difference in median age at diagnosis, 59 years for CWBI versus 63 years for HWBI (P = .004), and in median follow-up time, 97 months for CWBI versus 55 months for HWBI (P < .001). After accounting for differences in patient and tumor characteristics with inverse probability of treatment weighting, we found similar 5-year freedom from local recurrence (HR, 0.76; 95% CI, 0.14-4.1), freedom from regional recurrence (HR, 3.395% CI 0.15-69), freedom from distant metastasis (HR 3.9, 95% CI 0.86-17), and disease-free survival (HR 0.84; 95% CI, 0.3-2.4), between those treated with CWBI and those treated with HWBI. Results were similar among each of the 3 high-risk subtypes. ConclusionsOur data support the use of moderate HWBI in patients with early-stage, biologically high-risk breast cancer.

Abstract 4105: External user testing of Biofidelity’s ASPYRE-Lung assay demonstrates breakthrough capabilities and ease of use

Abstract Introduction: While the FDA has approved over 20 targeted therapies for non-small cell lung cancer (NSCLC), less than half of patients in the US undergo comprehensive biomarker testing to determine if they would benefit from these agents. This gap in testing is compounded in ex-US and minority populations. There are multiple barriers to access for biomarker testing, many of which stem from the complexity and high costs of current solutions, which limit testing to large, centralized laboratories. Alongside this, tissue requirements are high, turnaround times (TAT) for results typically take weeks, and reimbursement is uncertain. We demonstrate how ASPYRE can address these limitations by enabling ultra-sensitive biomarker testing using existing staff and widely available instrumentation. The technology is based on four enzyme steps (Silva et al 2020), the last of which is monitored on a real-time PCR instrument. ASPYRE-Lung targets 111 markers in 11 genes, including 77 DNA variants (substitutions and indels), and 34 RNA variants (fusions and exon skipping) across 24 wells, with a TAT of less than &amp;lt;4 hours from extracted nucleic acids to result. Experimental procedures: Four test sites were provided with ASPYRE reagents and contrived samples including Seracare ctDNA Mutation Mix v2 and a panel consisting of synthetic RNA oligonucleotides diluted in a background of normal lung RNA. The Seracare sample included variants in BRAF, EGFR, ERBB2, and KRAS at 0.25% variant allele fraction. The RNA sample included fusions in RET, ROS1 and NTRK1, with each fusion present at a mean of 6 copies in a background of 1 ng lung RNA. Operators from a range of educational backgrounds were provided a set of written Instructions for Use, with no additional training. Summary of data: The ASPYRE-Lung test was set up and performed at each test laboratory in as little as one day, using their existing real-time PCR instrument. The results demonstrated detection of the expected variants with consistent performance characteristics across the test sites and between users. Importantly, the sensitivity of detection was in line with, or superior to, the current gold standard. Conclusions: Biofidelity’s breakthrough ASPYRE technology enables a dramatic simplification of workflows, and the ultra-sensitive detection of actionable biomarkers using existing real-time PCR instruments. This work has demonstrated the ease with which independent laboratories can implement comprehensive multi-gene assays based on ASPYRE technology, utilizing their existing staff and infrastructure. This stands in stark contrast to alternative solutions, which typically require substantial investments in new instrumentation, skilled staff, IT infrastructure and bioinformatics. Taken together, ASPYRE promises to remove key barriers in cancer biomarker testing, enabling all patients to access the right treatment at the right time. Citation Format: Paulina Powalowska, Nicola Potts, Brandon A. Smith, Kala F. Schilter, Honey V. Reddi, Lan Beppu, Jerald Radich, Charles Massie, Quentin Vicentini, Tom Brown, Robert Osborne, Barnaby Balmforth. External user testing of Biofidelity’s ASPYRE-Lung assay demonstrates breakthrough capabilities and ease of use [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 4105.