Gene Expression Profile Test Research Articles

Analytical validity of DecisionDx-SCC, a gene expression profile test to identify risk of metastasis in cutaneous squamous cell carcinoma (SCC) patients

BackgroundTo improve identification of patients with cutaneous squamous cell carcinoma (SCC) at high risk for metastatic disease, the DecisionDx-SCC assay, a prognostic 40-gene expression profile (40-GEP) test, was developed and validated. The 40-GEP assay utilizes RT-PCR gene expression analysis on primary tumor biopsy tissue to evaluate the expression of 34 signature gene targets and 6 normalization genes. The test provides classifications of low risk (Class 1), moderate risk (Class 2A), and high risk (Class 2B) of metastasis within 3 years of diagnosis. The primary objective of this study was to validate the analytical performance of the 40-gene expression signature.MethodsThe repeatability and reproducibility of the 40-GEP test was evaluated by performance of inter-assay, intra-assay, and inter-operator precision experiments along with monitoring the reliability of sample and reagent stability for class call concordance. The technical performance of clinical orders from September 2020 through July 2021 for the 40-GEP test was assessed.ResultsPatient hematoxylin and eosin (H&E) stained slides were reviewed by a board-certified pathologist to assess minimum acceptable tumor content. Class specific controls (Class 1 and Class 2B) were evaluated with Levey-Jennings analysis and demonstrated consistent and reproducible results. Inter-assay, inter-operator and intra-assay concordance were all ≥90%, with short-term and long-term RNA stability also meeting minimum concordance requirements. Of the 2586 orders received, 93.5% remained eligible for testing, with 97.1% of all tested samples demonstrating actionable class call results.ConclusionDecisionDx-SCC demonstrates a high degree of analytical precision, yielding high concordance rates across multiple performance experiments, along with exhibiting robust technical reliability on clinical samples.

Using a 31-Gene Expression Profile Test to Stratify Patients with Stage I-II Cutaneous Melanoma According to Recurrence Risk: Update to a Prospective, Multicenter Study.

Simple SummaryMany people with skin cancer will have their cancer come back. The 31-gene expression profile (31-GEP) test can help predict if a cancer has a low (Class 1) or high (Class 2) chance of returning. This study looked at 86 patients with early skin cancer to see how well the 31-GEP test predicted if their cancer would return. None of the patients with a Class 1 GEP result had their cancer return within 3 years, but one-fourth of patients with a Class 2 result did. This study showed that the 31-GEP test can help predict if a patient’s skin cancer will return. Accurate risk prediction can help doctors make better treatment plans for patients with skin cancer.Background: Fifteen to forty percent of patients with localized cutaneous melanoma (CM) (stages I–II) will experience disease relapse. The 31-gene expression profile (31-GEP) uses gene expression data from the primary tumor in conjunction with clinicopathologic features to refine patient prognosis. The study’s objective was to evaluate 31-GEP risk stratification for disease-free survival (DFS) in a previously published cohort with longer follow-up. Methods: Patients with stage IB–II CM (n = 86) were prospectively tested with the 31-GEP. Follow-up time increased from 2.2 to 3.9 years. Patient outcomes were compared using Kaplan-Meier and Cox regression analysis. Results: A Class 2B result was a significant predictor of 3-year DFS (hazard ratio (HR) 8.4, p = 0.008) in univariate analysis. The 31-GEP significantly stratified patients by risk of relapse (p = 0.005). A Class 2B result was associated with a lower 3-year DFS (75.0%) than a Class 1A result (100%). The 31-GEP had a high sensitivity (77.8%) and negative predictive value (95.0%). Conclusions: The 31-GEP is a significant predictor of disease relapse in patients with stage IB–II melanoma and accurately stratified patients by risk of relapse.

Abstract P5-15-04: An intervention to help improve understanding of gene expression profiling tests in breast cancer: which is best, an information film or leaflet?

Abstract Background: Discussions between oncologists and patients about Gene Expression Profiling (GEP) risk of recurrence results can be challenging especially if scores are intermediate or close to the high or low risk of recurrence thresholds. Although many clinical teams provide patients with information leaflets, these often employ complex language written to meet regulatory guidelines rather than to educate effectively the end-user. We designed two 8-minute information films that were conversational in style with simple explanatory graphics. We then compared participants’ knowledge retention after reading leaflets explaining GEP tests and recurrence risks with that following their viewing of a film. Methods: Using a randomised factorial crossover design, at time-point one (T1) women aged 45-75 yrs without breast cancer, read either a leaflet about Oncotype DX or Prosigna testing or watched the corresponding information film. They could read or watch as many times as they liked and take notes. In structured interviews that were recorded, we asked 9 different questions to determine participants’ knowledge. Next day (T2) information in the opposite format was provided and knowledge was re-assessed. The maximum possible score was 18. Additional questions probed which modality participants had found the easiest to understand, their preferences for either the film or leaflet and reasons for these. Results: 120 women from different socio-educational groups took part, 60 of whom had Oncotype DX films and leaflets and 60 the Prosigna versions. Irrespective of the GEP test being described, their knowledge scores at T1 were significantly higher following viewing of the films compared with those after reading leaflets (mean difference 4.1 points; p<0.0001; 95% CI 3.2, 5.0). When participants read leaflets first and subsequently viewed films mean knowledge significantly increased (5.3 points, p<0.0001; 95% CI 4.4, 6.3). In contrast mean knowledge decreased by 2 points (p<0.01; 95% CI -3.1, -0.8) at T2 if the film was viewed first. Extraneous and sometimes irrelevant information already read in the leaflets had confused many participants. Overall clear preferences for the film were expressed (88/120; 73.3%); (leaflet = 28/120; 23.3%); 4/120; (3.3%) no preference. Primary reasons for this included the clarity, ease of understanding and the reassurance gained from the voice-overs in the films. Conclusion: Provision of complex information in a film format using simple graphics helped participants’ understanding of GEP test results more than reading information alone and was preferred significantly by the majority. Citation Format: Lesley Fallowfield, Daniel Farewell, Hywel Jones, Shirley F May, Susan L Catt, Rachel ML Starkings, Lucy Matthews, Valerie A Jenkins. An intervention to help improve understanding of gene expression profiling tests in breast cancer: which is best, an information film or leaflet? [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-15-04.

Abstract P1-08-18: Impact of gene expression profile testing for lymph node positive (LN+) , hormone receptor positive (HR+), HER2 negative (HER2-) breast cancer (BC) patients on the use of adjuvant chemotherapy in a large community cancer center

Abstract Background: Use of gene expression profiling (GEP) is considered standard of care, in deciding use of adjuvant chemotherapy in patients with LN negative breast cancer. However, there are limited data for the use of GEP in LN+ disease. Both Mammaprint™ and Oncotype Dx 21 gene recurrence score™ (RS) have been evaluated in LN+ disease, the data are not as robust as in the LN- population. Based on data from the RXPONDER trial and the MINDACT trial, the NCCN has recently recommended the use of Oncotype Dx to help decide the utility and benefit of adjuvant chemotherapy after resection in LN- , Hormone receptor Positive (HR+), HER2 negative (HER2-) breast cancer patients. Because of the potential major impact of these data on patient management and healthcare utilization, we decided to explore the uptake and implementation of this testing at a large community cancer center in order to assess and report real world data pertaining to this novel standard of care. Methods: Database search of the Electronic Medical Records used at Ironwood Cancer Center, revealed 2455 newly diagnosed patients with early stage breast cancer during the period from 1/1/2018 till 6/1/2021. HIPAA deidentified data was extracted with inclusion criteria of newly diagnosed women with LN+ (1-3 nodes), HR+, and Her2- which yielded 403 patients. Demographics & treatment data: Characteristics included menopausal status (Premenopausal 23%, Post Menopausal 77%), Surgery type (mastectomy 44%, breast conservation surgery 56%), size of primary tumor (0-1 cm 16 %, 1-2 cm 28%, 2-5 cm 43%, and > 5 cm 13%), LNs resected (Range 0-37, Median 4), # of LNs +(1-60%, 2-25%, 3-15%), and Tumor Grade (Grade 1-23%, 2-56%, 3-21%). GEP was performed in 62% of the patients. 49% received adjuvant chemotherapy (61% anthracycline based, 39% non anthracycline based). Of the patients tested, 41% received adjuvant chemotherapy. Adjuvant antihormonal therapy included aromatase inhibitors in 86% and Tamoxifen in 14%. 81% of the pre-menopausal patients received complete ovarian suppression. Results: Logistical Regression analysis in a non linear fashion, R2 data and Chi-square analysis was used to test the statistical significance of the observed relationship with respect to the expected relationship. The data was divided into 6 monthly intervals to allow comparison of the uptake of the test and impact on adjuvant chemotherapy decision making. There was a significant increase in GEP profile testing percentages when these intervals were compared across the years (R2 =0.69). Increase in GEP profiling was associated with lower utilization of adjuvant chemotherapy (R2= 0.84). There was a non-significant decline in the use of adjuvant chemotherapy (Chi2 NS, p value 0.22). Menopausal status, grade/size of tumor did not meet the statistical significance for the likelihood of utilization of the test. Currently multivariate analysis is being performed to examine the interplay between the numerous clinical factors on which the data was collected. This analysis will allow us to decide if we can longitudinally explore recurrence rates in each of these subsets. Conclusion: GEP has seen a significant uptake to help with adjuvant chemotherapy decision making in LN + (1-3) patients in the community cancer center setting over the last 4 years. These real world data also showed that increased testing was associated with decreased chemotherapy use which has translated to lowering health care resources and minimizing patient morbidity. Recommendations from the expert guidelines and clinical trial data have helped accelerate the use of this technology in the decision making to undergo adjuvant chemotherapy in the LN+, HR+, HER2- patients with breast cancer. Citation Format: Nisha Rao Kalmadi, Andrew Brown, Manas Sharma, Mikhail Shtivelband, Joshua Rifkind, Sujith Kalmadi, Rajesh Bagai, Emily Ho, Patricia Clark, Christopher Kellogg, Parvinderjit Khanuja. Impact of gene expression profile testing for lymph node positive (LN+) , hormone receptor positive (HR+), HER2 negative (HER2-) breast cancer (BC) patients on the use of adjuvant chemotherapy in a large community cancer center [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-18.

Expanded evidence that the 31-gene expression profile test provides clinical utility for melanoma management in a multicenter study

Objective National Comprehensive Cancer Network (NCCN) guidelines for cutaneous melanoma (CM) recommend physicians consider increased surveillance for patients who typically have lower melanoma survival rates (stages IIB-IV as determined by the American Joint Committee on Cancer (AJCC), 8th edition). However, up to 15% of patients identified as having a low recurrence risk (stages I-IIA) experience disease recurrence, and some patients identified as having a high recurrence risk will not experience any recurrence. The 31-gene expression profile test (31-GEP) stratifies patient recurrence risk into low (Class 1) and high (Class 2) and has demonstrated risk-appropriate impact on disease management and clinical decisions. Methods Five-year plans for lab work, frequency of clinical visits, and imaging pre- and post-31-GEP test results were assessed for a cohort of 509 stage I-III patients following an interim subset analysis of 247 patients. Results After receiving 31-GEP results, 50.6% of patients had a change in management plans in at least one of the following categories—clinical visits, lab work, or surveillance imaging. The changes aligned with the risk predicted by the 31-GEP for 76.1% of patients with a Class 1 result and 78.7% of patients with a Class 2 result. A Class 1 31-GEP result was associated with changes toward low-intensity management recommendations, while a Class 2 result was associated with changes toward high-intensity management recommendations. Conclusion The 31-GEP can stratify patient recurrence risk in patients with CM, and clinicians understand and apply the prognostic ability of the 31-GEP test to alter patient management in risk-appropriate directions.

Sparing the Prod: Providing an Alternative to Endomyocardial Biopsies With Noninvasive Surveillance After Heart Transplantation During COVID-19

BackgroundThe COVID-19 pandemic has reduced access to endomyocardial biopsy (EMB) rejection surveillance in heart transplant (HT) recipients. This study is the first in Canada to assess the role for noninvasive rejection surveillance in personalizing titration of immunosuppression and patient satisfaction post-HT. MethodsIn this mixed-methods prospective cohort study, adult HT recipients more than 6 months from HT had their routine EMBs replaced by noninvasive rejection surveillance with gene expression profiling (GEP) and donor-derived cell-free DNA (dd-cfDNA) testing. Demographics, outcomes of noninvasive surveillance score, hospital admissions, patient satisfaction, and health status on the medical outcomes study 12-item short-form health survey (SF-12) were collected and analyzed, using t tests and χ2 tests. Thematic qualitative analysis was performed for open-ended responses. ResultsAmong 90 patients, 31 (33%) were enrolled. A total of 36 combined GEP/dd-cfDNA tests were performed; 22 (61%) had negative results for both, 10 (27%) had positive GEP/negative dd-cfDNA results, 4 (11%) had negative GEP/positive dd-cfDNA results, and 0 were positive on both. All patients with a positive dd-cfDNA result (range: 0.19%-0.81%) underwent EMB with no significant cellular or antibody-mediated rejection. A total of 15 cases (42%) had immunosuppression reduction, and this increased to 55% in patients with negative concordant testing. Overall, patients’ reported satisfaction was 90%, and on thematic analysis they were more satisfied, with less anxiety, during the noninvasive testing experience. ConclusionsNoninvasive rejection surveillance was associated with the ability to lower immunosuppression, increase satisfaction, and reduce anxiety in HT recipients, minimizing exposure for patients and providers during a global pandemic.

Improved cutaneous melanoma survival stratification through integration of 31-gene expression profile testing with the American Joint Committee on Cancer 8th Edition Staging.

Cutaneous melanoma (CM) survival is assessed using averaged data from the American Joint Committee on Cancer 8th edition (AJCC8). However, subsets of AJCC8 stages I-III have better or worse survival than the predicted average value. The objective of this study was to determine if the 31-gene expression profile (31-GEP) test for CM can further risk-stratify melanoma-specific mortality within each AJCC8 stage. This retrospective multicenter study of 901 archival CM samples obtained from patients with stages I-III CM assessed 31-GEP test predictions of 5-year melanoma-specific survival (MSS) using Kaplan-Meier and Cox proportional hazards. In stage I-III CM population, patients with a Class 2B result had a lower 5-year MSS (77.8%) than patients with a Class 1A result (98.7%) and log-rank testing demonstrated significant stratification of MSS [χ2 (2df, n = 901) = 99.7, P < 0.001). Within each stage, 31-GEP data provided additional risk stratification, including in stage I [χ2 (2df, n = 415) = 11.3, P = 0.004]. Cox regression multivariable analysis showed that the 31-GEP test was a significant predictor of melanoma-specific mortality (MSM) in patients with stage I-III CM [hazard ratio: 6.44 (95% confidence interval: 2.61-15.85), P < 0.001]. This retrospective study focuses on Class 1A versus Class 2B results. Intermediate results (Class 1B/2A) comprised 21.6% of cases with survival rates between Class 1A and 2B, and similar to 5-year MSS AJCC stage values. Data from the 31-GEP test significantly differentiates MSM into lower (Class 1A) and higher risk (Class 2B) groups within each AJCC8 stage. Incorporating 31-GEP results into AJCC8 survival calculations has the potential to more precisely assess survival and enhance management guidance.

Gene Expression Profiling in Early Breast Cancer-Patient Stratification Based on Molecular and Tumor Microenvironment Features.

Patients with early-stage hormone receptor-positive, human epidermal growth factor receptor 2-negative (HER2−) breast cancer (BC) are typically treated with surgery, followed by adjuvant systemic endocrine therapy with or without adjuvant chemotherapy and radiation therapy. Current guidelines regarding the use of adjuvant systemic therapy depend on clinical and pathological factors, such as the morphological assessment of tumor subtype; histological grade; tumor size; lymphovascular invasion; and lymph node status combined with estrogen receptor, progesterone receptor, and HER2 biomarker profiles assessed using immunohistochemistry and in situ hybridization. Additionally, the prognostic and predictive value of tumor-infiltrating lymphocytes and their composition is emerging as a key marker in triple negative (TNBC) and HER2-enriched molecular breast tumor subtypes. However, all these factors do not necessarily reflect the molecular heterogeneity and complexity of breast cancer. In the last two decades, gene expression signatures or profiling (GEP) tests have been developed to predict the risk of disease recurrence and estimate the potential benefit of receiving adjuvant systemic chemotherapy in patients with luminal breast cancer. GEPs have been utilized to help physicians to refine decision-making process, complementing clinicopathological parameters, and can now be used to classify the risk of recurrence and tailoring personalized treatments. Several clinical trials using GEPs validate the increasing value of such assays in different clinical settings, addressing relevant clinical endpoints. Finally, the recent approval of immune checkpoint inhibitors in TNBC and the increasing use of immunotherapy in different molecular BC populations highlight the opportunity to refine current GEPs by including a variety of immune-related genes that may help to improve predicting drug response and finetune prognosis.

IMPARTER, Phase 1 of an intervention to improve patients\u2019 understanding of gene expression profiling tests in breast cancer

PurposeTo compare participants’ knowledge about gene expression profiling (GEP) tests and recurrence risks after reading an information leaflet with that following viewing of an information film.MethodsUsing a randomised cross-over design, at time-point one (T1), women aged 45–75 years without breast cancer either read leaflets or watched information films about Oncotype DX or Prosigna tests. Participants answered nine questions assessing knowledge (maximum score 18). Next-day information in the opposite modality was provided and knowledge re-assessed. Additional questions probed which format was easiest to understand, participants’ preferences for film or leaflet and their reasons for these.Results120 women participated (60 received OncotypeDX films and leaflets; 60 received the Prosigna versions). T1 mean knowledge scores were higher following film viewing (13.37) compared with that after reading leaflets (9.25) (mean difference 4.1; p < 0.0001; 95% CI 3.2, 5.0). When participants read leaflets first and subsequently viewed films, all increased their scores (mean + 6.08, from T1 of 9.25, p < 0.0001; 95% CI 5.44, 6.72). When films were viewed first, followed by leaflets, (36/60, 60%), participants’ scores declined (mean-1.55 from T1 of 13.37, p < 0.001; 95% CI -2.32, -0.78). A majority of participants expressed preferences for the films (88/120, 73.3%) irrespective as to whether they described OncotypeDX or Prosigna. Reasons included the clarity, ease of understanding, visual material and reassuring voice-over.ConclusionDiscussions between oncologists and patients about recurrence risk results can be challenging. Information leaflets may aid understanding but often employ complex language. Information films significantly improved knowledge and were preferred by participants.

Development and validation of a nomogram incorporating gene expression profiling and clinical factors for accurate prediction of metastasis in patients with cutaneous melanoma following Mohs micrographic surgery

There is a need to improve prognostic accuracy for patients with cutaneous melanoma. A 31-gene expression profile (31-GEP) test uses the molecular biology of primary tumors to identify individual patient metastatic risk. Develop a nomogram incorporating 31-GEP with relevant clinical factors to improve prognostic accuracy. In an IRB-approved study, 1124 patients from 9 Mohs micrographic surgery centers were prospectively enrolled, treated with Mohs micrographic surgery, and underwent 31-GEP testing. Data from 684 of those patients with at least 1-year follow-up or a metastatic event were included in nomogram development to predict metastatic risk. Logistic regression modeling of 31-GEP results and T stage provided the simplest nomogram with the lowest Bayesian information criteria score. Validation in an archival cohort (n=901) demonstrated a significant linear correlation between observed and nomogram-predicted risk of metastasis. The resulting nomogram more accurately predicts the risk for cutaneous melanoma metastasis than T stage or 31-GEP alone. The patient population is representative of Mohs micrographic surgery centers. Sentinel lymph node biopsy was not performed for most patients and could not be used in the nomogram. Integration of 31-GEP and T stage can gain clinically useful prognostic information from data obtained noninvasively.

Evolving Experience with TruGraf® Gene Expression Profile and TRAC™ Donor-Derived Cell-Free DNA Testing in Kidney Transplantation: First Year Post Transplant and Beyond

Evolving Experience with TruGraf® Gene Expression Profile and TRAC™ Donor-Derived Cell-Free DNA Testing in Kidney Transplantation: First Year Post Transplant and Beyond

The 31-gene expression profile stratifies recurrence and metastasis risk in patients with cutaneous melanoma.

Aim: Sentinel node biopsy is a prognostic indicator of melanoma recurrence. We hypothesized that adding the primary melanoma molecular signature from the 31-gene expression profile (31-GEP) test could refine the risk of recurrence prognosis for patients with stage I-III melanoma. Materials & methods: Four hundred thirty-eight patients with stage I-III melanoma consecutively tested with the 31-GEP were retrospectively analyzed. The 31-GEP stratified patients as low-risk (Class 1A), intermediate-risk (Class 1B/2A)or highrisk (Class 2B) of recurrence or metastasis. Results: The 31-GEP significantly stratified patient risk for recurrence-free survival (p<0.001), distant metastasis-free survival (p<0.001)and melanoma-specific survival (p<0.001) and was a significant, independent predictor of metastatic recurrence (hazard ratio: 5.38;p= 0.014). Conclusion: The 31-GEP improves prognostic accuracy in stage I-III melanoma.

Identifying Clinicopathological Factors Associated with Oncotype DX® 21-Gene Recurrence Score: A Real-World Retrospective Cohort Study of Breast Cancer Patients in Quebec City, Canada.

Gene expression profiling tests such as the Oncotype DX (ODX) 21-gene recurrence score (RS) assay is increasingly used in clinical practice to predict the risk of recurrence and support treatment planning for early-stage breast cancer (BC). However, this test has some disadvantages such as a high cost and a long turnaround time to get results, which may lead to disparities in access. We aim to identify clinicopathological factors associated with ODX RS in women with early-stage BC. We conducted a retrospective cohort study of women identified in the medical database of the Deschênes-Fabia Breast Disease Center of Quebec City University, Canada. Our sample consists of 425 women diagnosed with early-stage BC who have obtained an ODX RS between January 2011 and April 2015. The ODX RS has been categorized into three levels as originally defined: low (0–17), intermediate (18–30), and high (>30). The mean RS was 17.8 (SD = 9.2). Univariate analyses and multinomial logistic regressions were performed to identify factors associated with intermediate and high RS compared with low RS. A total of 237 (55.8%) patients had low RS, 148 (34.8%) had intermediate RS, and 40 (9.4%) had high RS. Women with progesterone receptor (PR)-negative (ORs ranging from 3.51 to 10.34) and histologic grade II (ORs ranging from 3.16 to 23.04) tumors were consistently more likely to have intermediate or high RS than low RS. Similar patterns of associations were observed when the RS was categorised using redefined thresholds from (i.e., from the TAILORx study or dichotomized). This study provides evidence suggesting that histologic grade and PR status are predictive factors for intermediate or high RS in women with early-stage BC. If these results are confirmed in future studies, considering these clinicopathological factors could spare women the need to get such a test before the beginning of a possible adjuvant therapy. This option could be considered in settings where the cost of testing is an issue.

Gene Expression Profiling (GEP) As a Prognostic Indicator of Multiple Myeloma, Irrespective of Disease Stage, Prior Treatment and Prior Transplant - a Single Center Experience

BackgroundMultiple myeloma (MM) risk is classified using fluorescence in situ hybridization (FISH) routine cytogenetics, and gene expression profiling (GEP). GEP is a clinically validated tool for prognostication of MM in newly diagnosed and relapsed patients (pts), its role in very heavily treated stages is less clear. A study (Nishihori et al, ASH, 2016) concluded that GEP is useful regardless of disease stage. We aim to determine the prognostic significance of GEP heavily pre-treated MM cohort.Methods299 pts with MyPRSTM GEP results at Hackensack University Medical Center (NJ) between 2012 and 2016 were included. High risk GEP was defined with the validated cutoff score of >45.2, high risk FISH was determined according to the mSMART classification as del17p, t(14,16), t(14,20). Chi-square tests were performed on categorical data and Wilcoxon rank sum tests on continuous data. Overall survival (OS) and progression free survival (PFS) were performed using Kaplan-Meier.ResultsMedian age was 63 years (range, 25-91), 170 pts (56%) were men, 81 pts (28%) had International Staging System (ISS) 3 and 191 pts (62%) had Durie-Salmon Staging (DSS) III. 245 pts (81%) had at least one bone marrow transplant (BMT). Median time from myeloma diagnosis to GEP was 30.1 (range 0.1-178.4) months. Most pts had GEP on diagnosis but 24% were heavily pretreated (range, 1-6 lines of treatment), and 13% had a BMT prior to GEP testing. 77 pts (25%) had high-risk disease based on GEP with a median score of 36.5 (range, 9.03-84.05). Molecular subtypes were hyperdiploidy (HY) 31%, cyclin family (CD)-2 20%, low bone disease (LB) 13%, proliferation (PR) 11%, MAF associated (MF) 10%, CD-1 8%, and MMSET associated (MS) 6%. Median OS and PFS were significantly inferior in patients with high-risk by GEP, 25 vs 33 months (p<0.021), 13 vs 21 months (p<0.004) respectively. Molecular subtypes were not individual predictors of OS (p<0.2). When MF, MS and PR subtypes were combined, they had inferior OS compared to other subtypes but this was not statistically significant (p<0.3).ConclusionsGEP can accurately predict risk status in heavily pre-treated and post-transplant MM pts. In conclusion, our data confirm that GEP is a good prognostic indicator in MM pts irrespective of disease stage and prior treatments. Further prospective studies with larger samples and correlating with baseline and intra-treatment GEP are needed. DisclosuresRichter:Janssen: Speakers Bureau; BMS: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vesole:Takeda: Speakers Bureau; Celgene: Speakers Bureau. Siegel:Celgene, Takeda, Amgen Inc, Novartis and BMS: Consultancy, Speakers Bureau; Merck: Consultancy. Biran:Takeda: Speakers Bureau; Celgene, Amgen: Consultancy, Speakers Bureau.

Clinical Considerations for Integrating Gene Expression Profiling into Cutaneous Squamous Cell Carcinoma Management.

Gene expression profile (GEP) testing is now commercially available for metastatic risk prediction in patients with cutaneous squamous cell carcinoma (CSCC) and one or more high-risk factors. The purpose of this article is to provide an early framework for healthcare providers looking to integrate patient-specific tumor biology into their clinical practice using GEP testing. To develop a framework for clinical use, an expert panel was convened to identify CSCC management decision points where GEP testing may be immediately incorporated into practice until the definitive results of prospective trials become available. Based on their discussion, the expert panel focused on the areas of nodal evaluation, adjuvant radiation therapy, and follow-up and surveillance. The panel emphasized that GEP prognostic test results should not currently be used as a surrogate for standard of care treatment but as an additional data point when determining individualized management for patients with high-risk CSCC. Whenever possible, decisions on management plans for these patients should be developed with multidisciplinary input. J Drugs Dermatol. 2021;20:6(Suppl):s5-11. doi:10.36849/JDD.6068.

ESCALADE: A phase 3 study of acalabrutinib in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for patients ≤65y with untreated non-germinal center B-cell–like (non-GCB) diffuse large B-cell lymphoma (DLBCL).

TPS7572 Background: R-CHOP remains the standard of care for DLBCL. Although most patients (pts) can be cured, 35–40% will experience relapsed/refractory disease, leading to poor outcomes in the majority of pts. Covalent irreversible Bruton tyrosine kinase inhibitors (BTKi) have shown higher responses in pts with non-GCB DLBCL than with GCB DLBCL. In untreated non-GCB DLBCL pts, the phase 3 PHOENIX study (Younes et al. J Clin Oncol. 2019;37:1285-95) showed that addition of the BTKi ibrutinib to R-CHOP (R-CHOP-I) did not improve outcomes in the intent-to-treat population. However, pts age &lt; 60y treated with R-CHOP-I had significantly improved progression-free survival (PFS) and overall survival (OS) compared with those receiving R-CHOP alone. Acalabrutinib (A) is a second-generation BTKi with enhanced kinase selectivity and potential for better efficacy and tolerability than first-generation BTKis. There is a strong rationale for combining A with R-CHOP in pts with untreated DLBCL, and safety of A + R-CHOP has been shown in a phase 1b/2 study (Davies et al. ASH 2020). The aim of this study is to determine if the addition of A to R-CHOP leads to improved PFS in pts age ≤65y with untreated non-GCB DLBCL. Methods: ESCALADE (ACE-LY-312; NCT04529772) is a phase 3, randomized, global, double-blind study of A vs placebo in combination with R-CHOP for treatment of newly diagnosed non-GCB DLBCL. The study is recruiting adults ≥18y and ≤65y with previously untreated DLBCL stage II–IV disease with a Revised International Prognostic Index (R-IPI) score of 2–5. Prior to randomization, all pts will receive an initial R-CHOP cycle (cycle 1) as standard-of-care treatment to prevent delays in therapy initiation. Based on central Gene Expression Profile (GEP) testing performed after enrollment, pts with non-GCB DLBCL (activated B-cell like or unclassified) will be randomized into 2 arms to receive A 100 mg twice daily plus R-CHOP or placebo plus R-CHOP from cycle 2 to cycle 6 followed by 2 additional cycles of rituximab + A or placebo (cycles 7 and 8). All pts will receive primary prophylaxis with granulocyte colony-stimulating factors accompanying all R-CHOP cycles. The study aims to randomize 600 pts (̃300 per arm). The primary objective is to evaluate whether the addition of A to R-CHOP will prolong PFS. Secondary endpoints include event-free survival, complete response rate, OS, pharmacokinetics, and safety. Key exclusion criteria are central nervous system involvement, primary mediastinal lymphoma, high-grade B-cell lymphoma, diagnosis or treatment of malignancy other than DLBCL, and history of indolent lymphoma. Approximately 250 sites globally will enroll pts. Enrollment began in Q3 of 2020. Clinical trial information: NCT04529772.

Long-Term Outcomes in a Multicenter, Prospective Cohort Evaluating the Prognostic 31-Gene Expression Profile for Cutaneous Melanoma.

PURPOSECurrent guidelines for postoperative management of patients with stage I-IIA cutaneous melanoma (CM) do not recommend routine cross-sectional imaging, yet many of these patients develop metastases. Methods that complement American Joint Committee on Cancer (AJCC) staging are needed to improve identification and treatment of these patients. A 31-gene expression profile (31-GEP) test predicts metastatic risk as low (class 1) or high (class 2). Prospective analysis of CM outcomes was performed to test the hypotheses that the 31-GEP provides prognostic value for patients with stage I-III CM, and that patients with stage I-IIA melanoma and class 2 31-GEP results have metastatic risk similar to patients for whom surveillance is recommended.MATERIALS AND METHODSTwo multicenter registry studies, INTEGRATE (ClinicalTrials.gov identifier:NCT02355574) and EXPAND (ClinicalTrials.gov identifier:NCT02355587), were initiated under institutional review board approval, and 323 patients with stage I-III CM and median follow-up time of 3.2 years met inclusion criteria. Primary end points were 3-year recurrence-free survival (RFS), distant metastasis-free survival (DMFS), and overall survival (OS).RESULTSThe 31-GEP was significant for RFS, DMFS, and OS in a univariate analysis and was a significant, independent predictor of RFS, DMFS, and OS in a multivariable analysis. GEP class 2 results were significantly associated with lower 3-year RFS, DMFS, and OS in all patients and those with stage I-IIA disease. Patients with stage I-IIA CM and a class 2 result had recurrence, distant metastasis, and death rates similar to patients with stage IIB-III CM. Combining 31-GEP results and AJCC staging enhanced sensitivity over each approach alone.CONCLUSIONThese data provide a rationale for using the 31-GEP along with AJCC staging, and suggest that patients with stage I-IIA CM and a class 2 31-GEP signature may be candidates for more intense follow-up.

Racial Disparities in Gene Expression Profiling but Not Donor-Derived Cell-Free DNA after Heart Transplant

<h3>Purpose</h3> Recipient race remains a powerful risk factor for mortality after heart transplantation (HT), with African Americans (AA) having worse survival than Caucasians. The aim of this analysis was to evaluate racial differences in gene expression profiling (GEP) and donor derived cell free DNA (dd-cfDNA) after HT using the Surveillance HeartCare Outcomes Registry (SHORE). <h3>Methods</h3> SHORE is an ongoing registry with >1200 patients enrolled at 60 HT centers in the United States. The registry includes HT recipients aged ≥15 years and ≥55 days post-transplant who had routine GEP and dd-cfDNA testing for rejection surveillance. Since patient sampling is from multiple centers, a linear mixed effects model, an extension of the linear model, was used to accommodate both fixed and random effects in the data. <h3>Results</h3> Among 956 recipients included in the analysis, 198 (20.7%) were AA, median age 57.0 years [0, 74.0] and 73.9% male. Overall, median GEP values demonstrated a borderline difference (p=0.07, Kolmogorov Smirnov test) for AA versus non-AA patients (Figure). This is evident by the cumulative distribution function, where a slightly higher rise in slope is observed for the non-AA cohort. Using linear mixed effects models, there was a statistically significant difference in GEP score when grouped by months post-transplant, demonstrating an early difference between AA and non-AA patients between months 2-5, but not subsequently. For dd-cfDNA analysis, median values were similar between AA (0.12%; Range 0.1-3.9%) and non-AA (0.12%; 0.1-3.5%) patients within the first year post-transplant (p=0.87, 2-sided K-S test) (Figure). <h3>Conclusion</h3> AA race is associated with higher GEP scores early post-transplant. This may be related to differential expression of the MARCH8 and FLT3 genes following HT, as previously described. Further analysis of specific GEP clusters and covariates across racial groups may shed light on these observed differences. In contrast, dd-cfDNA values do not appear to be influenced by race.

Longitudinal Variation in Absolute and Relative AlloMap Score in Heart Transplant Recipients

<h3>Purpose</h3> The non-invasive AlloMap gene expression profiling test has been demonstrated to possess high negative predictive value for detecting cardiac allograft rejection and dysfunction after heart transplant. We assessed longitudinal trends in AlloMap score post-transplant. <h3>Methods</h3> We retrospectively analyzed AlloMap scores, organized by season and year post-transplant, for patients undergoing heart transplantation at our center. Patients with multiple tests in one season had their scores averaged. Relative AlloMap score was calculated for each patient as (AlloMap score)/(average AlloMap score across all tests). The association between season and AlloMap score was also investigated. Seasons were defined as the following: (1) Winter: January - March, (2) Spring: April - June, (3) Summer: July - September, (4) Fall: October - December. <h3>Results</h3> A total of 658 tests were included in our study. AlloMap scores were significantly greater in tests done more than one-year post-transplant compared to those completed in the first year (median: 32 (29, 34) vs 28 (24.5, 32), p <0.0001). Similarly, the relative AlloMap score increased over time, observed to be significantly higher in tests performed after one-year compared to those done in the first year (median: 1.07 (1.01, 1.14) vs 0.97 (0.875, 1.02), p <0.0001). There was a trend towards increased AlloMap score in winter and fall, but this was not significant (p = 0.089). <h3>Conclusion</h3> We observed longitudinal trends of rising absolute and relative AlloMap scores after one-year post-transplant. Clinically, the variability score may be more meaningful than the raw score in the long-term due to more specific comparison of patient level variation from baseline. Further analyses with multicenter data are warranted.

A human stem cell-derived test system for agents modifying neuronal N-methyl-D-aspartate-type glutamate receptor Ca2+-signalling.

Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate “mixed cortical cultures” (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-d-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.