Primary Tumor Material Research Articles

Validation of a breast cancer assay for radiotherapy omission: an individual participant data meta-analysis.

There are currently no molecular tests to identify individual breast cancers where radiotherapy (RT) offers no benefit. Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) is a 16-gene molecular signature developed to identify low risk cancers where RT will not further reduce recurrence rates. An individual participant data meta-analysis was performed in 623 cases of node-negative ER+/HER2-negative early breast cancer enrolled in three RT randomized trials for whom primary tumor material was available for analysis. A Cox proportional hazards model on time to locoregional recurrence (LRR) was used to test the interaction between POLAR score and RT. 429 (69%) patients' tumors had a high POLAR score and 194 (31%) had a low score. Patients with high POLAR score had, in the absence of RT, a 10-year cumulative incidence of LRR: 20% (15%-26%) vs 5% (2%-11%) for those with a low score. Patients with a high POLAR score had a large benefit from RT (hazard ratio [HR] for RT vs no RT: 0.37 [0.23-0.60], p < .001). In contrast, there was no evidence of benefit from RT for patients with a low POLAR score (HR: 0.92 [0.42-2.02], p = .832). The test for interaction between RT and POLAR was statistically significant (p = .022). POLAR is not only prognostic for locoregional recurrence but also predictive of benefit from radiotherapy in selected patients. Patients ≥ 50 years with ER+/HER2-negative disease and a low POLAR score could consider omitting adjuvant RT. Further validation in contemporary clinical cohorts is required.

Relationship between infiltration of tissue-resident memory T cells (TRM) in head and neck squamous cell carcinoma (HNSCC) tissue and localization and age.

6089 Background: Elevated levels of tumor-infiltrating lymphocytes (TILs) have been correlated with improved survival rates in cancer patients. Among TIL subgroups, tissue-resident memory T cells (TRM, CD8+CD103+) are recognized as pivotal contributors to the anti-cancer immune response. In this study, we aimed to evaluate TRM presence in Head and Neck Squamous Cell Carcinoma (HNSCC) tissue utilizing a developed tissue microarray (TMA) and a multiplex immunohistochemistry (MxIHC) approach. Methods: HNSCC cases from Southampton Hospital spanning the years 2000 to 2016 were reviewed, resulting in the identification of approximately 300 cases with sufficient primary tumor material. A TMA was constructed by triplicate coring of marked tumor areas on all slides. Subsequently, MxIHC, incorporating markers such as CD8 and CD103, was performed using a stain-scan-strip methodology. Digital image analysis software was employed to analyze scanned slides, and a quality check (QC) was conducted. Results: Following QC, 193 primary tumors remained (hypopharynx and larynx: n = 28, lip and oral cavity: n = 51, oropharynx: n = 114). Significantly higher counts of CD8 T cells and TRM were observed in the combined group of oropharynx, lip and oral cavity compared to the combined group of larynx and hypopharynx. This significance persisted even after excluding HPV negative oropharynx cases. HNSCC of the lip and oral cavity itself had statistically more TRM than larynx and hypopharynx. No differences were found in the subgroups between tonsil and tongue base. Stratifying the analysis by age revealed a statistically significant increase in TRM infiltration in HNSCC among young patients (≤50 years) compared to the 51-60 years and 61-70 years age groups, a pattern not observed for CD8 alone. Conclusions: This study underscores the importance of HNSCC heterogeneity and highlights the impact of age on immune infiltration, emphasizing the unique contribution of TRM in the anti-cancer immune response.

Abstract 235: Application of high-grade serous ovarian cancer organoids in functional precision medicine

Abstract High-grade serous ovarian cancer (HGSC) is the most frequent and lethal ovarian cancer type. Despite responding favorably to first-line therapy, most HGSC patients eventually relapse. HGSC is characterized by high interpatient genomic heterogeneity and few recurrent mutations that could be used as therapeutic biomarkers. Thus, except the introduction of PARP inhibitors for BRCA1/2-mutated patients, there has been little progress with personalizing HGSC treatment. Functional precision medicine - tailoring personalized therapies based on functional assays on patient-derived cells - has been applied in cancer clinical trials to address this issue. Organoids - three-dimensional cell cultures, recapitulating aspects of original tumors’ structure and function - have often been used as models in these efforts. However, inefficient and time-consuming organoid derivation protocols have been hindering the application of HGSC organoids in functional precision medicine clinical trials, due to the short time during which the therapy must be selected. To address this, we here aimed to establish whether we could predict second-line therapy drug sensitivities in relapsed HGSC using organoids from tumor material from first-line therapy. We have previously derived a collection of 23 organoid models from 16 patients, sampled sequentially at different times of their treatment (1). Here, for several organoid primary-relapse pairs, we performed drug screens using a library of 370 compounds (approved, in clinical development or tool compounds). We found that 90% of the hit compounds in the primary organoids also were hits in the matched relapse-derived organoid models. In several patients, we observed a pronounced cytotoxicity of several cyclin-dependent kinase (CDK) inhibitors, all of which potently inhibit CDK9, including two selective inhibitors (AZD4573 and VIP152) in clinical development. Screening of CDK9 inhibitors in an extended panel of organoid models revealed that most organoids are sensitive to CDK9 inhibition in nanomolar range. CDK9 inhibitors also prevented organoid outgrowth in long-term culture (&amp;gt;1.5 month) in drug-free medium. Taken together, we demonstrate that HGSC organoids from material sampled during first-line treatment predict most drug sensitivities of relapse-derived organoids. This suggests a feasibility of functional precision approach for selection of second-line therapy using only primary tumor material, circumventing the problem of limited time available for the therapy choice after tumor sampling. This work also highlights that CDK9 inhibitors, currently in clinical development for hematological malignancies, are worthwhile to be explored in HGSC. 1. Senkowski et al. A platform for efficient establishment and drug-response profiling of high-grade serous ovarian cancer organoids. Developmental Cell (2023) Citation Format: Wojciech Senkowski, Laura Gall-Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Aura Crispino, Jaana Oikkonen, Daria Bulanova, Yan-Jun Chen, Elena M. Doncel, Karolin Voßgröne, Erdogan P. Erkan, Jun Dai, Anastasia Lundgren, Jutta Huvila, Kaisa Huhtinen, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. Application of high-grade serous ovarian cancer organoids in functional precision medicine [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 235.

Abstract 5309: ATOR-4066, a Neo-X-Prime™ bispecific antibody targeting CD40 and CEACAM5, induces tumor localized immune cell activation in preclinical in vivo tumor model

Abstract ATOR-4066 is a preclinical stage bispecific antibody targeting CD40 and CEACAM5, based on Alligator’s novel Neo-X-Prime™ antibody technology platform. ATOR-4066 induces a neoantigen specific T-cell response by simultaneous binding to CD40 on myeloid cells such as antigen presenting cells (APCs) and to CEACAM5 on tumor cells. CEACAM5 is a tumor-associated antigen highly expressed on several cancer indications such as colorectal and gastric cancer, as well as on tumor-derived material. ATOR-4066 treatment results in (1) CEACAM5-conditional CD40-mediated activation of APCs and (2) enhanced uptake of tumor-derived material, leading to cross-presentation of neoantigen and priming of neoantigen-specific T cells, resulting in enhanced tumor cell killing. We have previously shown evidence of a potent anti-tumor effect of ATOR-4066 in vivo, superior to the effects observed with the corresponding CD40 mAb. Moreover, we have demonstrated CEACAM5-dependent activation of CD40-expressing cells in vitro, and an ability to mediate co-localization of CEACAM5-expressing tumor debris and CD40 expressing APCs. In addition, ATOR-4066 was shown to induce CEACAM5-conditional activation of tumor-infiltrating CD40-expressing immune cells in dissociated primary human tumor material from colorectal and gastric cancer patients. Here we present data further elucidating the mode of action of ATOR-4066 in vivo. Following ATOR-4066 treatment, tumors and lymph nodes were isolated from human CD40 transgenic mice bearing MC38 tumors expressing human CEACAM5. Flow cytometry analysis shows an increased number of tumor-infiltrating immune cells in mice treated with ATOR-4066 compared to treatments with CD40 mAb or vehicle. The study also reveals upregulation of co-stimulatory molecules such as CD86 on DCs and macrophages after treatment with ATOR-4066 compared to groups treated with CD40 mAb or vehicle. The effect was localized to the tumor microenvironment and not observed in tumor draining lymph nodes, confirming previous in vitro results demonstrating CEACAM5-conditional immune activation. Moreover, RNA-sequencing of tumor samples displayed distinct transcriptomic alterations following ATOR-4066 treatment vs. CD40 mAb or vehicle treatment in vivo. In contrast, RNA-sequencing of peripheral blood in ATOR-4066 treated mice showed only small transcriptomic differences compared to the vehicle treated group, but distinct from mice treated with a CD40 mAb, further demonstrating the CEACAM5 dependent response of ATOR-4066. In summary, these preclinical data demonstrate that ATOR-4066 activates infiltrating myeloid cells resulting in increases in activated tumor infiltrating T cells, thus creating a pro-inflammatory tumor microenvironment, which strongly supports further development and clinical testing of ATOR-4066. Citation Format: Hampus Andersson, Ida Uddbäck, Mona Celander, Maria Johansson, Lill Ljung, Anneli Nilsson, Amulya Krishna Shetty, David Gomez Jimenez, Mattias Levin, Sara Fritzell, Laura von Schantz, Malin Lindstedt, Karin Hägerbrand, Anette Sundstedt, Peter Ellmark. ATOR-4066, a Neo-X-Prime™ bispecific antibody targeting CD40 and CEACAM5, induces tumor localized immune cell activation in preclinical in vivo tumor model [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 5309.

Abstract 4258: An innovative PDX and patient-derived 3D platform for improved translatability of new medical entities

Abstract The preclinical platform landscape in oncology has radically changed in the last 15 years, mainly due to an increased ability to generate patient-derived models. Multiple 3D tumor cell cultures methods, either from established cell lines or from primary cultures, have been developed to assess the activity of therapeutic agents in vitro. These methods are strongly influenced by the experimental conditions, such as cell culture conditions, the presence of 3D supports like Matrigel, and the experimental design of the assay, such as the choice of the dose and the duration of the assay. The ability of these methods to predict drug efficacy in the patient relies not only in the optimization of the experimental settings, but also on the representativeness of the patient population, and on the prevention of model drifts due to clonal selection and/or tumor cell adaptation to culture conditions with passages. In this project, we aimed to develop an innovative 3D cell model platform that addresses all these experimental challenges. To do so, we took advantage of our low passage Tumorgraft platform, a bank of more than 1500 patient-derived xenografts (PDXs) well characterized at molecular and pharmacological level and highly representative of the patient population, to generate the Tumorgraft3D platform. We derived TumorGraft3D models from low passage PDXs, by using proprietary, indication-specific culture conditions. By keeping these models at low passage, we were able to avoid clonal drifts. Moreover, the use of indication specific media allowed the preservation of patient’s and PDX’s tumor histology, molecular traits at genetic, gene expression and proteomic level, and displayed proliferation rates that recapitulate those observed in the parental PDXs. So far, we have successfully developed over 100 models from breast, ovarian, prostate, NSCLC, colorectal and gastric indications, with success rates up to 90%. Ex-vivo testing of standard of care highlighted different response profiles in accordance with the molecular feature of these models. The tumor dissociation process and culture conditions optimized with PDXs were also used for the development of 3D cultures derived from patients’ samples. These data demonstrated that our 3D platform can integrate the complementarity of our PDX and patient-derived 3D platform, which lies in the possibility of evaluating test agent activity in the repeatable and reproducible setting provided by the patient-derived 3D models, as well as in models directly derived from patients with more heterogeneous and indication-specific establishment success rate, and throughput dependent on the amount of primary tumor material. Citation Format: Samaneh Kamali, Stefano Cairo, Fu-Ju Chou, Taylor Light, Mara Gilardi, Brandon Walling, Christine Baer, Hsiu-Wen Tsai, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. An innovative PDX and patient-derived 3D platform for improved translatability of new medical entities [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 4258.

Identification of a ΔNp63-Dependent Basal-Like A Subtype-Specific Transcribed Enhancer Program (B-STEP) in Aggressive Pancreatic Ductal Adenocarcinoma.

Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

Abstract GS4-03: Validation of Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) in a meta-analysis of three randomized controlled trials of breast conserving surgery +/- radiotherapy

Abstract Background: There are currently no commercially available tests to identify early stage breast cancer patients treated with breast conserving surgery (BCS) and systemic therapy at low risk of locoregional recurrence (LRR) for whom postoperative radiotherapy (RT) may be safely omitted. Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) is a 16-gene molecular signature developed to identify invasive breast cancer patients who may be candidates for RT omission after BCS. In this work, we seek to validate POLAR in a meta-analysis of three RCTs of BCS +/- RT: SweBCG91RT, Scottish Conservation Trial (SCT) and Princess Margaret Hospital (PMH). Methods: A patient-level meta-analysis was performed in 623 node-negative breast cancer patients with ER+/HER2-negative tumors who were enrolled in the three RCTs and for whom primary tumor material was available for analysis. Contributions from each cohort were as follows: SweBCG91RT N=354 (57%), SCT N=137 (22%), and PMH N=132 (21%). Numbers of LRR events in each cohort were as follows: SweBCG91RT N=72 (20%), SCT N=28 (20%), and PMH N=16 (12%). There was a mix of systemic therapy used (no systemic therapy for SweBCG91RT, chemotherapy or adjuvant endocrine therapy, but not both, in SCT, and tamoxifen but no chemotherapy for PMH). Median follow-up time for the patients who did not have LRR was 13.3 years for SweBCG91RT, 21.1 years for SCT, and 8.6 years for PMH. A multivariable Cox proportional hazards model on time to LRR, including the continuous standardized POLAR score, RT, and interaction, stratified by cohort, was used to test the interaction between the continuous POLAR score and RT. Additional Cox models tested the association between treatment arms separately for patients with a low and high POLAR score using a pre-specified cut point. Cumulative incidences were computed, with distant metastasis and death without recurrence considered as competing events. Results: The test for interaction between RT treatment and POLAR was statistically significant (p = 0.022). Patients with a high POLAR score (N=429 [69%]) had a large benefit from RT (10-year cumulative incidence of LRR: 20% [15%-26%] for those not treated with RT vs 7% [4%-11%] for those treated with RT; hazard ratio for RT vs no RT: 0.37 [0.23-0.60], p &amp;lt; 0.001), whereas there was no evidence of benefit from RT for patients with a low POLAR score (N=194 [31%], 10-year cumulative incidence of LRR: 5% [2%-11%] for those not treated with RT vs 7% [3%-14%] for those treated with RT; hazard ratio for RT vs no RT: 0.92 [0.42-2.02], p = 0.832). Conclusions: To our knowledge, POLAR is the first genomic classifier that is not only prognostic for LRR but also predictive, showing a significant interaction between RT and the classifier. Patients with a high POLAR score should be recommended radiotherapy while patients with a low score may be candidates for omission of radiotherapy after breast conserving surgery. Citation Format: Per Karlsson, Anthony Fyles, S. Laura Chang, Bradley Arrick, Frederick Baehner, Per Malmström, Mårten Fernö, Erik Holmberg, Martin Sjöström, Fei-Fei Liu, David A. Cameron, Linda J. Williams, John MS Bartlett, Joanna Dunlop, Jacqueline Caldwell, Joseph F. Loane, Elizabeth Mallon, Tammy Piper, Wilma J. Jack, Ian Kunkler, Felix Y. Feng, Corey W. Speers, Lori Pierce, John Bennett, Karen J. Taylor. Validation of Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) in a meta-analysis of three randomized controlled trials of breast conserving surgery +/- radiotherapy [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 GS4-03.

Establishing a Microfluidic Tumor Slice Culture Platform to Study Drug Response.

Accurate models for tumor biology and prediction of drug responses of individual tumors require novel technology to grow tumor tissue ex vivo to maintain tumor growth characteristics in situ. Models containing only tumor cells, without the stromal components of the tumor, are suboptimal for many purposes and are generally problematic because the cells are passed through extensive culture and selection. Therefore, direct culture of (human) tumors is of considerable interest for basic tumor biology and diagnostic purposes. Microfluidic technologies have been proposed to accurately mimic physiological conditions for tissue growth. Most published systems build tissues from individual cell types in so-called Organ-on-Chip (OoC) cultures. We here describe a novel OoC device for growing tumor specimens. Thin tumor slices are grown in a microfluidic 'chip' that allows precisely controlled in vitro culture conditions. The performance of the OoC device was extensively validated for predicting therapeutic responses in human breast cancer patient-derived xenograft (PDX) tumor material. The system is amenable to primary tumor material from surgery or biopsies. In addition to using the model to predict and evaluate therapeutic responses, the model can also be used for mechanistic studies of human cancers, such as clonal evolution or immune responses, or to validate new or repurposed (cancer) drugs. The Bi/ond Cancer-on-Chip (CoC) device is designed to culture tumor slices and investigate aspects of tumor growth and drug responses. Here, we describe the step-by-step process of setting up tumor slice cultures using a Bi/ond CoC device and performing in vitro drug response evaluation. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of breast cancer tumor slice culture using a microfluidic cancer-on-chip platform for chemotherapy testing ex vivo Basic Protocol 2: Histology and immunohistochemistry-based analysis of tumor tissue architecture, cell proliferation, and cell death.

Spatial immunophenotypes of distant metastases and response to immune checkpoint inhibition.

538 Background: The value of PD-L1 to predict durable responses to immune checkpoint inhibition (ICI) in metastatic urothelial carcinoma (mUC) is inconsistent. We hypothesize that the use of archived primary tumor material (PRIM) for PD-L1 testing in clinical trials not properly reflecting the metastatic disease status (MET) contributes to this clinical issue. Objective: To analyze the predictive and prognostic value of PD-L1, spatial immunephenotypes and MHC-I determined in patient-matched PRIM/MET. Methods: PD-L1 (Ventana IC-Score, combined positivity score), spatial immunephenotypes (midi-plex digital spatial immuneprofiling) and MHC-I were examined in 154 mUC patients with at least one available pretreatment MET (138 patient-matched PRIM/MET pairs). 119 patients received first-line platinum-based chemotherapy, and 50 patients received second-line immunecheckpoint inhibition. PD-L1 expression, spatial immunephenotypes and MHC-I status of (patient-matched PRIM and) pretreatment MET were correlated to chemotherapy and ICI response and outcomes. Results: Discordance rates in patient-matched PRIM/MET amounted 25/30%, 36% and 49% for PD-L1 (CPS10/IC5%), immunephenotypes and MHC-I (loss versus preserved), respectively. Correlations with chemotherapy and ICI responses were observed for immunephenotypes and MHC-I status determined in MET (not for PD-L1 alone), but not in PRIM. In case of ICI, patients with cytotoxic tumor immune microenvironments (TIME) showed durable responses with disease control rates of 90% and a hazard ratio for disease progression/death of 0.05 (95%-CI:0.01-0.65) versus patients with immunedepleted MET (DCR 29%). MET MHC-I status added incremental value to predict durable ICI responses: Combination of MHC-I based (auto-)antigen expression of tumor cells with spatial immunepehnotypes in pre-treatment MET improved predictive and prognostic impact for response and outcome prediction of mUC patients undergoing first-line platinum-based chemotherapy and second-line immunecheckpoint inhibition. Limitations include the partly retrospective design and the lack of MET multisampling on individual patient level. Conclusions: The TIME is subject to substantial dynamics during metastatic evolution. MET immunephenotypes and MHC-I statuses show promising potential to predict chemotherapy and durable ICI responses, while the PRIM TIME does not. Thus, future clinical trials should rather rely on pre-treatment MET-biopsies reflecting the current immunological disease state than on PRIM.

Spatial Immunephenotypes of Distant Metastases but not Matched Primary Urothelial Carcinomas Predict Response to Immune Checkpoint Inhibition

BackgroundThe value of programmed cell death ligand-1 (PD-L1) to predict durable responses to immune checkpoint inhibitors (ICIs) in metastatic urothelial carcinoma (mUC) is inconsistent. We hypothesize that the use of archived primary tumor material (PRIM) for PD-L1 testing in clinical trials not properly reflecting the metastatic disease status (MET) contributes to this clinical issue. ObjectiveTo analyze the predictive and prognostic value of PD-L1, spatial immunephenotypes, and major histocompatibility complex class I (MHC-I) determined in patient-matched PRIM/MET. Design, setting, and participantsPD-L1, spatial immunephenotypes, and MHC-I were examined in 154 mUC patients with at least one available pretreatment MET (138 patient-matched PRIM/MET pairs). Outcome measurements and statistical analysisPD-L1, spatial immunephenotype, and MHC-I status of (patient-matched PRIM and) pretreatment MET were correlated with chemotherapy and ICI response and outcomes. Results and limitationsDiscordance rates in patient-matched PRIM/MET were 25/30%, 36%, and 49% for PD-L1 (CPS10/IC5%), immunephenotypes, and MHC-I (loss vs preserved), respectively. Correlations with chemotherapy and ICI responses were observed for immunephenotypes and MHC-I status determined in MET (not for PD-L1 alone), but not in PRIM. In case of ICIs, patients with cytotoxic tumor immune microenvironment (TIME) showed durable responses with disease control rates of 90% and a hazard ratio for disease progression/death of 0.05 (95% confidence interval: 0.01–0.65) versus patients with immunedepleted MET (disease control rate 29%). MET MHC-I status added an incremental value to predict durable ICI responses. Limitations include the partly retrospective design and the lack of MET multisampling on individual patient level. ConclusionsThe TIME is subject to substantial dynamics during metastatic evolution. MET immunephenotypes and MHC-I statuses show promising potential to predict chemotherapy and durable ICI responses, while the PRIM TIME does not. Thus, future clinical trials should rather rely on pretreatment MET biopsies reflecting the current immunological disease state than on PRIM. Patient summaryPrediction of chemotherapy and responses to immune checkpoint inhibitors might be possible using representative pretreatment metastatic biopsies.

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

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

Distinct immune stimulatory effects of anti-human VISTA antibodies are determined by Fc-receptor interaction.

VISTA (PD-1H) is an immune regulatory molecule considered part of the next wave of immuno-oncology targets. VISTA is an immunoglobulin (Ig) superfamily cell surface molecule mainly expressed on myeloid cells, and to some extent on NK cells and T cells. In previous preclinical studies, some VISTA-targeting antibodies provided immune inhibitory signals, while other antibodies triggered immune stimulatory signals. Importantly, for therapeutic antibodies, the isotype backbone can have a strong impact on antibody function. To elucidate the mode of action of immune stimulatory anti-VISTA antibodies, we studied three different anti-human VISTA antibody clones, each on three different IgG isotypes currently used for therapeutic antibodies: unaltered IgG1 (IgG1-WT), IgG1-KO (IgG1-LL234,235AA-variant with reduced Fc-effector function), and IgG4-Pro (IgG4- S228P-variant with stabilized hinge region). Antibody functionality was analysed in mixed leukocyte reaction (MLR) of human peripheral blood mononuclear cells (PBMCs), as a model system for ongoing immune reactions, on unstimulated human PBMCs, as a model system for a resting immune system, and also on acute myeloid leukemia (AML) patient samples to evaluate anti-VISTA antibody effects on primary tumor material. The functions of three anti-human VISTA antibodies were determined by their IgG isotype backbones. An MLR of healthy donor PBMCs was effectively augmented by anti-VISTA-IgG4-Pro and anti-VISTA-IgG1-WT antibodies, as indicated by increased levels of cytokines, T cell activation markers and T cell proliferation. However, in a culture of unstimulated PBMCs of single healthy donors, only anti-VISTA-IgG1-WT antibodies increased the activation marker HLA-DR on resting myeloid cells, and chemokine levels. Interestingly, interactions with different Fc-receptors were required for these effects, namely CD64 for augmentation of MLR, and CD16 for activation of resting myeloid cells. Furthermore, anti-VISTA-IgG1-KO antibodies had nearly no impact in any model system. Similarly, in AML patient samples, anti-VISTA-antibody on IgG4-Pro backbone, but not on IgG1-KO backbone, increased interactions, as a novel readout of activity, between immune cells and CD34+ AML cancer cells. In conclusion, the immune stimulatory effects of antagonistic anti-VISTA antibodies are defined by the antibody isotype and interaction with different Fc-gamma-receptors, highlighting the importance of understanding these interactions when designing immune stimulatory antibody therapeutics for immuno-oncology applications.

Abstract 1700: Serial monitoring of single-cell circulating tumor cell genomics in metastatic lobular breast cancer to identify precision and immuno-oncology biomarkers with therapeutic implications

Abstract Clinical decisions for precision and immuno-oncology therapies are based on predictive biomarkers commonly obtained from a single metastatic biopsy, or from archived primary tumor material. Circulating genomic biomarkers present a minimally invasive way to monitor the intra-patient tumor heterogeneity and its fluctuations in order to provide a real-time evaluation of the changing clonal architecture with potential therapeutic implications. Single-cell DNA next generation sequencing (scNGS) of circulating tumor cells (CTC) is a particularly well-suited method of unraveling and monitoring that heterogeneity to complement biomarker information obtained from tissue and cell-free circulating tumor DNA (ctDNA). In this proof-of-concept study we analyzed 123 CTC, 15 white blood cells (WBC), and ctDNA from 15 CTC-positive lobular breast cancer patients, five of whom had CTC available at both metastatic baseline and after progression on a variety of therapies chosen at their physician’s discretion. CTC were enriched with the CellSearch® system and isolated as single cells with the DEPArray™ system. Whole genome amplified CTC DNA underwent scNGS with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space to detect mutations, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI). 99.1% of cells were informative, with a mean sequencing depth of 664x. Using our previously developed, CTC-based precision medicine reporting platform, MI-CTCSeq, multiple CTC in seven of 15 patients (47%) had mutations that were actionable by FDA-approved targeted therapies including in the oncogenes PIK3CA (alpelisib) and FGFR2 (erdafitinib). 13 patients (87%) displayed intra-patient, inter-CTC genomic heterogeneity of putative driver mutations. Two of five (40%) patients with CTC at both baseline and progression displayed fluctuations in their CTC subclonal makeup between timepoints. One of the two harbored a baseline ESR1 (estrogen receptor α) p.D538G activating mutation that largely disappeared at progression and was replaced by a CTC subclone with a different ESR1 activating mutation, p.Y537S. Intriguingly, this patient’s CTC also harbored an FGFR2 p.K659M mutation in an actionable “hotspot” at progression, which was absent at baseline, suggesting potential utility of serial monitoring by CTC scNGS. TMB scores and MSI status in CTC were highly concordant with those measured in clinical tissue biopsies. Taken together, these data suggest the non-invasive interrogation of the CTC genomic landscape and its serial monitoring to inform precision and immuno-oncology treatments in real time. Citation Format: Andi K. Cani, Emily M. Dolce, Elizabeth P. Darga, Kevin Hu, Chia-Jen Liu, James M. Rae, Daffyd G. Thomas, Scott A. Tomlins, Arul M. Chinnaiyan, Aaron M. Udager, Costanza Paoletti, Erin F. Cobain, Daniel F. Hayes. Serial monitoring of single-cell circulating tumor cell genomics in metastatic lobular breast cancer to identify precision and immuno-oncology biomarkers with therapeutic implications [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 1700.

Noncanonical Function of AGO2 Augments T-cell Receptor Signaling in T-cell Prolymphocytic Leukemia.

The identification of AGO2-mediated activation of oncogenic T cells through signal amplifying protein-protein interactions advances the understanding of leukemogenic AGO2 functions and underlines the role of aberrant TCR signaling in T-PLL.

The 3D in vivo chorioallantoic membrane model and its role in breast cancer research.

We aimed to evaluate the role of the chorioallantoic membrane model (CAM) in breast cancer research. The following is an overview of the use of the CAM in the field of breast cancer research based on a PubMed literature query. The CAM is a 3D in vivo model that can be used for the analysis of tumor growth, biology and angiogenesis of primary tumor tissue or tumor cell lines. The CAM model has been used in breast cancer research for drug testing, migration assays and the evaluation of vascularization, amongst others. The CAM model is a valuable method that offers a better imitation of the physiological phenomena compared to 2D or 3D in vitro models. The CAM model has primarily and successfully been utilized for the assessment of the tumor biology of established breast cancer cell lines. Further, the CAM model is a promising method to analyze patient derived primary tumor material and could be used as a "patient-specific 3D-tumor-therapy-model" for the cost-efficient evaluation of anti-cancer drugs to find the optimal treatment for breast cancer patients.

Relapsed Medulloblastoma in Pre-Irradiated Patients: Current Practice for Diagnostics and Treatment.

Simple SummaryMedulloblastoma is the commonest malignant brain tumour of childhood. Disease relapse following maximal multi-modal therapy including upfront craniospinal irradiation (CSI) is almost always fatal and occurs in approximately 30% of patients. Importantly, patients rarely die of other causes, and consequently relapsed medulloblastoma (rMB) accounts for 10% of all childhood cancer deaths. There is a variety of life-prolonging treatment options at relapse including neurosurgery, re-irradiation, early-phase trials, and chemotherapy. Here, we summarise best practice for investigations including re-biopsy and molecular characterisation of rMB, treatment decision making, and available treatment options. We also highlight advances in the understanding of rMB disease biology and prognostic factors, and look towards future developments such as targeted therapies, liquid biopsies and disease modelling strategies to improve outcomes in this area of unmet clinical need.Relapsed medulloblastoma (rMB) accounts for a considerable, and disproportionate amount of childhood cancer deaths. Recent advances have gone someway to characterising disease biology at relapse including second malignancies that often cannot be distinguished from relapse on imaging alone. Furthermore, there are now multiple international early-phase trials exploring drug–target matches across a range of high-risk/relapsed paediatric tumours. Despite these advances, treatment at relapse in pre-irradiated patients is typically non-curative and focuses on providing life-prolonging and symptom-modifying care that is tailored to the needs and wishes of the individual and their family. Here, we describe the current understanding of prognostic factors at disease relapse such as principal molecular group, adverse molecular biology, and timing of relapse. We provide an overview of the clinical diagnostic process including signs and symptoms, staging investigations, and molecular pathology, followed by a summary of treatment modalities and considerations. Finally, we summarise future directions to progress understanding of treatment resistance and the biological mechanisms underpinning early therapy-refractory and relapsed disease. These initiatives include development of comprehensive and collaborative molecular profiling approaches at relapse, liquid biopsies such as cerebrospinal fluid (CSF) as a biomarker of minimal residual disease (MRD), modelling strategies, and the use of primary tumour material for real-time drug screening approaches.

A Microfluidic Cancer-on-Chip Platform Predicts Drug Response Using Organotypic Tumor Slice Culture.

Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies. SIGNIFICANCE: The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.

Assessment of breast cancer primary tumor material in a 3D in vivo model.

Breast cancer is the most common malignant tumor in women and highly heterogeneous with a variety of different molecular subtypes. The analysis of the individual tumor biology is necessary to develop a specific and individualized treatment plan for every patient. The chick chorioallantoic membrane (CAM) model, a 3D-in-vivo-tumor-model, could potentially provide a methodology that facilitates the gain of additional information regarding the tumor biology as well as the testing of the tumor's individual sensitivity to different therapies. The objective was to establish the grafting of different breast cancer primaries onto the CAM for tumor profiling and the investigation of different parameters. Breast cancer primary tissue of different patients was grafted onto the CAM. Subsequently, 3D volume and perfusion measurements were performed during the engraftment period. Histological analyses of the tumors were carried out after the engraftment period. The grafting of the breast cancer primaries onto the CAM was successful. The tumors remained partially vital and displayed angiogenic development on the CAM. Breast cancer primary material can be grafted onto the CAM and we observed visible and measurable changes of perfusion over time.

Tumour-Derived Cell Lines and Their Potential for Therapy Prediction in Patients with Metastatic Colorectal Cancer.

Simple SummaryColorectal cancer is a global issue with millions of patients in need for new treatment options. Surgical resection is often the first clinical intervention and can be, especially for primary tumours, curative. However, advanced tumours, recurrences, and metastases require drug treatment. Our aim was to compare material of primary tumours and corresponding metastases to draw conclusions from the first one to the latter. The feasibility of therapy prediction using the primary tumour material for the metastatic situation creates a time window for functional testing, which can realistically be integrated into the strict timeframe of clinical procedures. A reliable treatment recommendation, based on the tumour drug response, would improve treatment success in patients with metachronous metastases and spare them from unnecessary side effects of unsuccessful therapies.The prognosis of metastatic colorectal cancer (CRC) remains poor. Patients and physicians are in need of individual therapies and precise response predictions. We investigated the predictive capacity of primary tumour material for treatment response of metastases. Mutational landscapes of primary tumours and corresponding metastases of 10 CRC patients were compared. Cell line characteristics and chemosensitivity were investigated pairwise for primary and metastatic tumours of four patients. PDX models of one patient were treated in vivo for proof of concept. Driver mutations did not differ between primaries and metastases, while the latter accumulated additional mutations. In vitro chemosensitivity testing revealed no differences for responses to 5-FU and oxaliplatin between primary and metastatic cell lines. However, irinotecan response differed significantly: the majority of metastases-derived cell lines was less sensitive to irinotecan than their matching primary counterpart. Therapy recommendations based on these findings were compared to clinical treatment response and mostly in line with the predicted outcome. Therefore, primary tumour cell models seem to be a good tool for drug response testing and conclusion drawing for later metastases. With further data from tumour-derived cell models, such predictions could improve clinical treatment decisions, both recommending likely effective therapeutic options while excluding ineffective treatments.

Integrated Analysis Of Immunotherapy Treated Clear Cell Renal Cell Carcinomas: An Exploratory Study.

Molecular or immunological differences between responders and nonresponders to immune checkpoint inhibitors (ICIs) of clear cell renal cell carcinomas (ccRCCs) remain incompletely understood. To address this question, we performed next-generation sequencing, methylation analysis, genome wide copy number analysis, targeted RNA sequencing and T-cell receptor sequencing, and we studied frequencies of tumor-infiltrating CD8+ T cells, presence of tertiary lymphoid structures (TLS) and PD-L1 expression in 8 treatment-naive ccRCC patients subsequently treated with ICI (3 responders, 5 nonresponders). Unexpectedly, we identified decreased frequencies of CD8+ tumor-infiltrating T cells and TLS, and a decreased expression of PD-L1 in ICI responders when compared with nonresponders. However, neither tumor-specific genetic alterations nor gene expression profiles correlated with response to ICI or the observed immune features. Our results underline the challenge to stratify ccRCC patients for immunotherapy based on routinely available pathologic primary tumor material, even with advanced technologies. Our findings emphasize the analysis of pretreated metastatic tissue in line with recent observations describing treatment effects on the tumor microenvironment. In addition, our data call for further investigation of additional parameters in a larger ccRCC cohort to understand the mechanistic implications of the observed differences in tumor-infiltrating CD8+ T cells, TLS, and PD-L1 expression.