Tumor-specific Mutations Research Articles

Systematic pan-cancer analysis of mutation-treatment interactions using large real-world clinicogenomics data.

Quantifying the effectiveness of different cancer therapies in patients with specific tumor mutations is critical for improving patient outcomes and advancing precision medicine. Here we perform a large-scale computational analysis of 40,903 US patients with cancer who have detailed mutation profiles, treatment sequences and outcomes derived from electronic health records. We systematically identify 458 mutations that predict the survival of patients on specific immunotherapies, chemotherapy agents or targeted therapies across eight common cancer types. We further characterize mutation-mutation interactions that impact the outcomes of targeted therapies. This work demonstrates how computational analysis of large real-world data generates insights, hypotheses and resources to enable precision oncology.

Detection and Quantification of ctDNA for Longitudinal Monitoring of Treatment in Non-Small Cell Lung Cancer Patients Using a Universal Mutant Detection Assay by Denaturing Capillary Electrophoresis.

Background: Observation of anticancer therapy effect by monitoring of minimal residual disease (MRD) is becoming an important tool in management of non-small cell lung cancer (NSCLC). The approach is based on periodic detection and quantification of tumor-specific somatic DNA mutation in circulating tumor DNA (ctDNA) extracted from patient plasma. For such repetitive testing, complex liquid-biopsy techniques relying on ultra-deep NGS sequencing are impractical. There are other, cost-effective, methods for ctDNA analysis, typically based on quantitative PCR or digital PCR, which are applicable for detecting specific individual mutations in hotspots. While such methods are routinely used in NSCLC therapy prediction, however, extension to cover broader spectrum of mutations (e.g., in tumor suppressor genes) is required for universal longitudinal MRD monitoring. Methods: For a set of tissue samples from 81 NSCLC patients we have applied a denaturing capillary electrophoresis (DCE) for initial detection of somatic mutations within 8 predesigned PCR amplicons covering oncogenes and tumor suppressor genes. Mutation-negative samples were then subjected to a large panel NGS sequencing. For each patient mutation found in tissue was then traced over time in ctDNA by DCE. Results: In total we have detected a somatic mutation in tissue of 63 patients. For those we have then prospectively analyzed ctDNA from collected plasma samples over a period of up to 2 years. The dynamics of ctDNA during the initial chemotherapy therapy cycles as well as in the long-term follow-up matched the clinically observed response. Conclusion: Detection and quantification of tumor-specific mutations in ctDNA represents a viable complement to MRD monitoring during therapy of NSCLC patients. The presented approach relying on initial tissue mutation detection by DCE combined with NGS and a subsequent ctDNA mutation testing by DCE only represents a cost-effective approach for its routine implementation.

Quantitative assessment of copy number alterations by liquid biopsy for neuroblastoma.

Liquid biopsy, a method of detecting genomic alterations using blood specimens, has recently attracted attention as a noninvasive alternative to surgical tissue biopsy. We attempted quantitative analysis to detect amplification of MYCN (MYCNamp) and loss of heterozygosity at 11q (11qLOH), which are clinical requisites as prognostic factors of neuroblastoma (NB). In this study, cell-free DNA (cfDNA) was extracted from plasma samples from 24 NB patients at diagnosis. Copy numbers of MYCN and NAGK genes were quantitatively analyzed by droplet digital PCR (ddPCR). 11qLOH was also assessed by detecting allelic imbalances of heterozygous single nucleotide polymorphisms in the 11q region. The results obtained were compared to those of specimens from tumor tissues. The correlation coefficient of MYCN copy number of cfDNA and tumor DNA was 0.88 (p < 0.00001). 11qLOH was also accurately detected from cfDNA, except for one case with localized NB. Given the high accuracy of liquid biopsy, to investigate components of cfDNA, the proportion of tumor-derived DNA was estimated by examining the variant allele frequency of tumor-specific mutations in cfDNA. The proportion of tumor-derived DNA in cfDNA was 42.5% (range, 16.9%-55.9%), suggesting sufficient sensitivity of liquid biopsy for NB. In conclusion, MYCN copy number and 11qLOH could be quantitatively analyzed in plasma cfDNA by ddPCR assay. These results suggest that plasma cfDNA can be substituted for tumor DNA and can also be applied for comprehensive genomic profiling analysis.

Abstract 2829: Non-viral precision genome engineering enables personalized adoptive neoTCR T cell therapy for cancer including multiple additional edits that improve the activity of neoTCR T cells by enhancing CD4 T cell antigen sensitivity and conferring resistance to TGFβ

Abstract Personalized autologous TCR-T cell therapies targeting neoepitopes (neoE) derived from tumor-specific mutations are a compelling approach for the treatment of patients with solid tumors. Using the ultra-sensitive imPACT Isolation Technology®, antigen-experienced, neoE-specific CD8 T cells are captured from the blood of patients with solid cancers followed by cloning of the cognate, neoE-specific, MHC class I-restricted T cell receptors (neoTCRs). DNA-mediated (non-viral) precision genome engineering technology is then used to engineer autologous CD8 and CD4 T cells to express the neoTCR. We have further built upon this platform to modify neoTCR-T cells to address potential sources for tumor immune evasion. The versatility of this single-step gene editing platform is demonstrated here by the presentation of a neoTCR T cell product with knockout of endogenous TCRα and TCRβ and the simultaneous expression of a neoTCR with CD8 co-receptor (CD8coR) from the same expression cassette along with knockout of TGFβ receptor 2 (TGFBR2). We have previously shown that CD8coR expression augments the activity of CD4 T cells engineered to express MHC class I restricted neoTCRs, by increasing CD4 neoTCR T cell helper and effector function. TGFβ is known to promote tumor growth, metastasis, and epithelial to mesenchymal transition (EMT) and to inhibit effector immune responses while promoting fibrosis and the differentiation of inhibitory cell types such as regulatory T cells. TGFβ is expressed by many tumor types and high TGFβ expression is associated with worse prognosis for several cancer subtypes including colorectal, lung and glioblastoma. TGFBR2 is a critical mediator of TGFβ signaling, resulting in potent, inhibitory effects on T cell function. Our data show that simultaneous ablation of TGFBR2 signaling and expression of CD8coR was achieved with high efficiency, resulting in fully functional CD8 and CD4 neoTCR-T cells. Deletion of TGFBR2 with or without CD8coR expression, together with the expression of a neoE-targeted TCR preserved T cell effector function in the presence of inhibitory concentrations of TGFβ. Importantly, the combination of TGFBR2 knockout and CD8coR expression resulted in additive benefits, providing proof-of-concept for modifying two orthogonal features to improve neoTCR T cell function. Altogether, these results demonstrate the applicability of this versatile, precision genome engineering platform technology to yield enhanced, next-generation neoTCR-T cell therapies to expand the potential for clinical benefit in persons with solid cancers. Citation Format: Charles W. Tran, Kayla Lee, Bhamini Purandare, James Byers, Michael M. Dubreuil, William Lu, Michal Mass, Kyle Jacoby, Stefanie J. Mandl, Barbara Sennino. Non-viral precision genome engineering enables personalized adoptive neoTCR T cell therapy for cancer including multiple additional edits that improve the activity of neoTCR T cells by enhancing CD4 T cell antigen sensitivity and conferring resistance to TGFβ [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 2829.

Abstract 253: Analyses of tumor-specific T cell dynamics and tumor immune contexture in microsatellite instability-high cancers in response to first-line therapy

Abstract Microsatellite instability-high (MSI-H) phenotype arises from defective DNA repair mechanisms and is found in many malignancies, including colorectal and endometrial cancers. Due to high neoantigen loads derived from tumor-specific mutations, evidence of tumor-infiltrating T cells and high response rates to checkpoint blockade (30-60%), MSI-H cancers can be leveraged to study the features of neoantigen-induced anti-tumor immunity in response to therapy and disease outcome. Previously we identified a set of recurrent frameshift (fs) mutations in MSI-H cancers that yielded immunogenic polyepitope neoantigens (neoAgs) eliciting CD8+ T cell responses. Here we investigate whether these shared fs-neoAgs engendered spontaneous T cell responses in MSI-H colorectal and endometrial cancer patients. Using peripheral blood mononuclear cells (PBMCs), we evaluated the frequency of the fs-neoAg-specific T cells by ex vivo IFN-γ ELISPOT upon fs-peptide stimulation. Additionally, we isolated naïve and memory T cells from MSI-H patient PBMCs and expanded fs-neoAg-specific T cells within each subset. Fs-neoAg-induced effector cytokine (IFN-γ, TNF-α) production was measured by flow cytometry and was detected only in the memory subset, supporting the hypothesis that shared fs-neoAgs prime CD8+ T cells in vivo to generate clonally-expanded memory T cells in MSI-H patients. To further evaluate the clonal expansion of fs-neoAg-specific T cells in MSI-H cancer patients, we identified fs-neoAg-specific T cell receptors (TCRs). T cell lines enriched for fs-neoAg-specific T cells were generated from patient PBMCs and sequenced for variable β chains of TCRs. These fs-neoAg-specific TCRs were monitored in the blood and tumor tissues, including those from primary and recurrent sites. Finally, to determine the spatial relationships between the infiltrating immune cells and the malignant cells at single cell resolution, we performed multiplexed immunohistochemistry using CODEX technology. A panel of 35 markers was used to evaluate the myeloid (HLA-DR, CD11c, CD14, CD68, CD206) and T (CD4, CD8, Foxp3) cell subsets, the activation (CD107a, CD69, Ki67) and exhaustion (PD-1, TIM3, LAG3) status of T cells, T cell states (TCF-1, TOX, PD-1) and the presence of tertiary lymphoid structures/antigen presenting cell niches (CD19, CD20, HLA-DR) in the tumor microenvironment of primary or recurrent MS-stable and MSI-H tumors from endometrial cancer patients. We also investigated potential immune escape mechanisms in MSI-H tumors, such as the loss of B2M and MHC-I expression. This study will provide insights into the quality and quantity of shared anti-tumor T cells within the periphery and tumor tissues and will help identify the immune correlates of response or resistance to therapy in MSI-H cancer patients. Citation Format: Cansu Cimen Bozkus, Mona Saleh, Rachel Brody, Stephanie V. Blank, Nina Bhardwaj. Analyses of tumor-specific T cell dynamics and tumor immune contexture in microsatellite instability-high cancers in response to first-line therapy [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 253.

Abstract 3574: A Phase I trial of personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients under Bruton-Tyrosin-Kinase-inhibitor based regimes - Preclinical development and study design

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Despite the therapeutic success of novel small molecules (e.g. ibrutinib, idealisib and venetoclax), patients frequently experience disease relapse due to the persistence of minimal residual disease (MRD). Targeting the residual CLL cells by immunotherapy could further improve the depth and persistence of CLL remission. Here, we report on the preclinical development and clinical implementation of a phase I trial evaluating safety, immunogenicity and efficacy in terms of MRD response of a personalized multi-peptide vaccine adjuvanted with the novel toll-like receptor (TLR) 1/2 agonist XS15 emulsified in Montanide࣪ ISA51 VG (Rammensee et al. JIC 2019) in CLL patient receiving Bruton-Tyrosin-Kinase (BTK)-inhibitor based regimes (NCT04688385). So far, broad application of peptide vaccines in cancer patients is hampered by time- and cost-intensive design and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. To improve on these drawbacks, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics (Nelde et al., Front Immunol, 2021). For the trial reported here, comparative mass spectrometry-based immunopeptidome analyses of 61 CLL patient samples and a dataset of benign tissues samples (n = 351) enabled the identification of high-frequent non-mutated CLL-associated antigens for the most common HLA allotypes (HLA-A*02, -A*24, and -B*07). These antigens were proven to be recognized as T cell epitopes by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, thereby enabling the selection of a 12-peptides panel comprising 9 HLA-class I (3 for each HLA allotype) and 3 HLA class II restricted high frequent CLL-exclusive T cell epitopes as warehouse for the personalized composition of study vaccine cocktails. Our meanwhile ongoing phase I trial recruits CLL patients eligible for treatment with BTK inhibitors. For those with matching HLA allotypes, a personalized peptide-cocktail is formulated comprising 6 CLL-associated peptides (3 HLA class I and 3 HLA class II peptides), selected from our warehouse based on individual immunopeptidome analysis, and 2 highly immunogenic HLA class II control peptides, one viral (human adenovirus C) and one tumor associated (Baculoviral IAP repeat-containing protein 5). Three doses of the vaccine are administered in a 4-weekly interval, after reduction of CLL-load by BTK inhibitors. So far, 10 CLL patients were included in the trial. The first vaccinations will be applied in November and December 2021 with first immunogenicity data expected in March 2022 and to be reported at the meeting. Citation Format: Maddalena Marconato, Annika Nelde, Yacine Maringer, Marcel Wacker, Marion Richter, Monika Denk, Helmut Salih, Jonas Heitmann, Juliane Sarah Walz. A Phase I trial of personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients under Bruton-Tyrosin-Kinase-inhibitor based regimes - Preclinical development and study design [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 3574.

Abstract 1375: The intra-tumoral spatial heterogeneity of T-cell antigens in glioblastoma: An integrated multi-omics approach

Abstract Glioblastoma is the most common primary malignant neoplasm of the central nervous system in adults. Current treatment options comprise maximal surgical resection followed by radiation and/or chemotherapy with temozolomide. However, these procedures are unable to eliminate all tumor cells, which in turn lead to disease recurrence accounting for the poor prognosis. Glioblastoma is a highly infiltrative tumor in which recurrence originates from the unresectable peritumoral infiltration zone. Thus, novel treatment options specifically targeting the tumor cells in the infiltration zone are needed to prevent relapse and enable long-lasting remission. In this work, we performed multi-omics spatial analysis of the necrotic center (NEC), the gadolinium contrast-enhanced region (T1), and the infiltration zone (INF) to assess immunological relevant aspects of tumor heterogeneity. By integrating mass spectrometry-based immunopeptidome analysis with next generation sequencing methods (whole exome and RNA sequencing, DNA methylation), we aimed to identify the intra-tumoral regional heterogeneity of T cell antigens with a special focus on the peritumoral infiltration zone. For multi-omics analysis, HLA peptides and genetic material from 15 glioblastoma patients were extracted and analyzed from the three zones NEC, T1, INF and for four patients additionally from adjacent benign (BEN) brain tissue. A total of 24,699 unique HLA class I and 17,394 HLA class II peptides were identified. Comparative profiling of peptides from our study and a benign tissue database (in-house (n=429) combined with HLA ligand atlas (https://hla-ligand-atlas.org)) revealed that 6% (970/15,579), 5% (829/16,442) and 5% (725/13,891) of HLA class I peptides were exclusively presented in the INF, T1 and NEC zone, respectively. 6% (394/7,150), 7% (589/8,736) and 13% (1,725/12,908) of HLA class II peptides showed exclusive representation in the INF, T1 and NEC zone, respectively. Importantly, due to the spatial analysis of the immunopeptidome, we revealed 970 HLA class I and 394 HLA class II peptides exclusively presented in the INF zone. Interestingly, one INF-associated HLA class I peptide, which showed frequent presentation in 36% of glioblastoma immunopeptidomes, is derived from the brain and acute leukemia cytoplasmic protein (BAALC), a highly expressed cell cycle inducer (via MEK kinase-1) in several cancers, including glioblastoma. Integrated RNA/DNA sequencing enabled a greater understanding of spatial tumor antigen presentation and lead to the identification of INF-specific neoepitopes derived from tumor-specific mutations. In summary, we identified the intra-tumoral regional heterogeneity of tumor antigens, which could be used in the future for specific immunotherapy approaches targeting the infiltration zone of glioblastoma. Citation Format: Marcel Wacker, Gioele Medici, Marissa Dubbelaar, Jens Bauer, Annika Nelde, Luca Regli, Michael Weller, Helmut R. Salih, Hans-Georg Rammensee, Marian C. Neidert, Juliane S. Walz. The intra-tumoral spatial heterogeneity of T-cell antigens in glioblastoma: An integrated multi-omics approach [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 1375.

Abstract 6388: The effect of oncolytic virus therapy on neoantigen specific immune responses

Abstract Background: Patients with malignant brain tumor, glioblastoma (GBM), do not benefit from promising immunotherapies that use checkpoint inhibitors, such as PD-1 and CTLA-4 blockade antibodies. Several reasons may underlie this clinical result, particularly because of immunogenically cold nature of the GBM microenvironment, which is characterized by the low level of infiltrating cytotoxic CD8+ T cells and by an enriched myeloid suppressor cell population. We and other groups hypothesize that oncolytic virus (OVs)-based treatments can overcome this problem. Our preclinical GBM models and an ongoing clinical trial with oncolytic herpes simplex virus-1 (oHSV-1) at our institution (NCT03152318) demonstrated that OV treatment leads to an increase in the influx of CD8+ and CD4+ T cells into TME. However, it remained unknown whether these T cells are specific to the tumor. Here, we evaluated T-cell responses against newly identified neoantigens from murine syngeneic glioma line in response to the intratumoral OV treatments. Results: To evaluate the effect of oHSV-1 treatment on neoantigen-specific responses, we first performed whole-exome and RNA sequencing of murine GBM for the identification of tumor-specific mutations. Putative MHC I antigens were predicted with netMHCpan V4 and immunogenicity of candidates, that were selected based on affinity to MHC I molecules, were checked by IFN-gamma enzyme-linked immunospot (ELISpot) assay. With our workflow, 91 H2-Db-restricted and 77 H2-Kb-restricted peptides were identified. From this group, 8 Db- and 9 Kb-restricted peptides were selected based on their highest MHC affinity. We then asked which of these neoantigens would show enhanced presentation upon oHSV infection in vivo. To determine that we first implanted GBM tumors in the brains of syngeneic mice. At Day7 post-tumor implantation, the oHSV-1 (or vehicle control), was in situ stereotactically administered. Seven days later, splenocytes, PBMCs, and TILs were harvested to be screened. TILs from the oHSV-1 treated mice, but not those from the control group reacted to the putative neoantigens, and this activation was similar in magnitude to the response to the immunodominant HSV peptide. We are currently performing immunopeptidome analyses to confirm the binding of these neoepitopes to MHC upon oHSV treatment and in vivo studies to determine how T cells specific to these MHC-I restricted neoepitopes contribute to the anti-tumor effect. Conclusion: Our data with the preclinical model of GBM show for the first time that infection with an oHSV-1 does indeed lead to novel TIL activation against tumor neoantigens that were not presented without OV infection. Thus, oncolytic viruses may offer an in-situ vaccination approach to stimulate neoantigen-specific immune responses. Citation Format: Raziye Piranlioglu, Junfeng Liu, Anan Islam, Alex Ling, Naoyuki Shono, William F. Goins, Hiroshi Nakashima, E. Antonio Chiocca. The effect of oncolytic virus therapy on neoantigen specific immune responses [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 6388.

Abstract 3555: Immunopeptidomics-guided tumor antigen warehouse design for peptide-based immunotherapy in head and neck squamous cell carcinomas

Abstract Head and neck squamous cell carcinomas (HNSCC) are the sixth most common malignancies worldwide. 45% of patients are diagnosed at a late tumor stage associated with poor survival. For metastatic, unresectable or recurrent (m/uR) HNSCC, immune checkpoint inhibition (ICI) was recently approved as a novel therapeutic option showing significant survival benefits compared to standard chemotherapy-based treatment. However, response to ICI is still limited to a small number of patients calling for further improvement of T cell-based immunotherapies. Peptide-based approaches, which rely on the specific immune recognition of tumor-associated human leukocyte antigen (HLA) presented peptides, represent promising and low side effect treatment options. Peptide vaccination has been shown to enhance and induce long-term anti-tumoral immune responses and even clinical responses in HNSCC patients. However, current vaccines are either monovalent, based on patient-individual tumor-specific mutations or restricted to a single HLA allotype and therefore neither widely applicable nor suitable for reliable studies and large-scale production. In this study, using mass spectrometry (MS) -based immunopeptidome analysis of a large cohort of HNSCC patient (n = 30) tumor and adjacent benign samples, we established a tumor-associated off-the-shelf peptide warehouse for broadly applicable personalized therapies. The malignant dataset, comprising 91651 HLA ligands, was compared to adjacent benign and various benign tissues (www.hla-ligand-atlas.org) to identify tumor-exclusive antigens. Further antigen selection was based on allotype-specific high frequent presentation. In total, 23 frequently presented and tumor-exclusive HNSCC-associated peptides were selected for six of the most common HLA class I allotypes (A*01, A*02, A*24, B*15, B*35, B*40) covering &amp;gt;75% of the world population, as well as five HLA class II presented peptides binding various different HLA class II allotypes. Immunogenicity was validated by IFN-γ ELISPOT screening for spontaneous preexisting T cell responses targeting the respective peptides as well as by in vitro priming experiments of naïve T cells in HNSCC patients and healthy volunteers. Furthermore, immunopeptidome analyses identified these antigens in patient plasma samples providing first evidence for “liquid biopsy” immunopeptidome analysis without the need of primary tumor tissue. A phase I study evaluating safety, immunogenicity as well as first efficacy of this warehouse-based vaccine in combination with ICI in HNSCC patients is currently being set up, with personalized peptide selection based on individual HLA-allotype and MS analysis of patient tumor/plasma sample. In conclusion, we here designed a peptide warehouse that enables a polyvalent and widely applicable but still personalized peptide vaccination in HNSCC patients. Citation Format: Sarah Schroeder, Thorben Gross, Annika Nelde, Marcel Wacker, Jens Bauer, Jonas Rieth, Marissa Dubbelaar, Lena Muehlenbruch, Yacine Maringer, Paul-Stefan Mauz, Martin Sailer, Julia Philipp, Sven Becker, Thomas Breuer, Helmut R. Salih, Hans-Georg Rammensee, Hubert Löwenheim, Juliane S. Walz. Immunopeptidomics-guided tumor antigen warehouse design for peptide-based immunotherapy in head and neck squamous cell carcinomas [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 3555.

Abstract 1991: Characterization of the exome, transcriptome, and immunopeptidome to map alterations in primary and recurrent glioblastoma

Abstract Glioblastoma is known as the most aggressive and most common malignant primary tumor in the central nervous system. Current treatment options comprise maximal surgical resection followed by radiation and/or chemotherapy with temozolomide. However, these therapies are not able to eliminate all tumor cells, which in turn inevitably leads to disease recurrence and an alteration of identified targets in the context of clonal evolution and potential hypermutation. T cell-based immunotherapy holds great promise to target malignant cells with CAR T cell and vaccination strategies, showing first promising results in glioblastoma. These therapies rely on the rejection of cancer cells through recognition of tumor antigens and T cell-mediated cytotoxicity. In previous work, we have characterized such tumor antigens in primary glioblastoma (Neidert et al., Acta Neuropathol, 2018), nonetheless, alterations in relapsed disease have not been addressed thus far. This study investigated the whole exome, transcriptome, and mass-spectrometry-based immunopeptidome of 38 primary and 24 recurrent tumors, including 22 autologous glioblastoma pairs, to determine alterations that occur during glioblastoma progression on multiple comics levels. In concordance with Neftel et al., Cell, 2019, we identified mutations that can be allocated to astrocyte- and mesenchymal-like classified genes. In addition, an increase in the mutation rate in recurrent glioblastoma was observed which is attributed to radiation and chemotherapy pretreatment of tumors. These newly arising tumor-specific mutations give rise to HLA-presented neoepitopes in the primary cohort. Moreover, we identified transcripts that are differentially expressed between the two cohorts, showing a higher expression of transcripts related to immune system responses in the recurrent cohort. Immunopeptidome analysis of the two cohorts revealed high frequent glioblastoma-exclusive HLA class I and class II ligands presented in both the primary and recurrent cohort, serving as universally applicable tumor antigens. Class I and II HLA ligands of each sample were analyzed and revealed 2,146 HLA class I- and 2,753 HLA class II presented antigens that were uniquely identified on primary glioblastoma. A total of 610 and 1,886 source proteins represent recurrence-exclusive antigens presented on HLA class I or II molecules, respectively. Together this work addressed differences in tumor antigen expression and presentation between primary and recurrent glioblastoma using these omics layers to create an overview of the alterations that occur during disease progression. Besides providing a deep insight into the glioblastoma (immuno-)biology during progression, this study yields targets for innovative immunotherapeutic approaches to eliminate residual cells and improve survival in glioblastoma patients. Citation Format: Marissa L. Dubbelaar, Lena K. Freudenmann, Jonas Scheid, Julia Velz, Gioele Medici, Konstantina Kapolou, Malte Mohme, Leon Bichmann, Marie Gauder, Stefan Czemmel, Christopher Mohr, Daniel J. Kowalewski, Manfred Westphal, Katrin Lamszus, Luca Regli, Michael Weller, Hans-Georg Rammensee, Helmut Salih, Marian C. Neidert, Juliane S. Walz. Characterization of the exome, transcriptome, and immunopeptidome to map alterations in primary and recurrent glioblastoma [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 1991.

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

Abstract Background: The analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision making and disease monitoring. A suitable diagnostic-grade platform is required for detection of tumor-specific mutations with high sensitivity in circulating cell-free DNA (ccfDNA) of cancer patients. Methods: In an international multicenter study conducted by three laboratories participating in the CANCER-ID consortium, a cohort containing 177 cell-free plasma samples at baseline, follow-up or disease progression derived from 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC), relevant mutations were tested using the UltraSEEK® Lung Panel on the MassARRAY® System covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2 and PIK3CA, and compared against mutation-specific droplet digital PCR (ddPCR) and tumor tissue next-generation sequencing (NGS) performed in routine diagnostics. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy were analyzed. Results: The UltraSEEK® Lung Panel revealed an overall detection rate of 87.2% irrespective of ccfDNA input, and a specificity of &amp;gt;99.5% using the reference material. When comparing the 131 variants identified in the patient-derived cell-free plasma using UltraSEEK® with mutation-specific ddPCR analyses, a concordance of 90% was found. The 77 diagnostically or clinically relevant variants identified in 66 pretreatment tumor tissue showed an overall concordance with UltraSEEK® of 73% at baseline with a false-negative rate of 3%. When restricting to therapeutically targetable mutations, the concordance elevated to 85%. A decrease in ctDNA levels at 4-6 weeks after start of treatment detected with UltraSEEK® correlated with a prolonged median PFS (46 vs 11 weeks; P&amp;lt;0.05) and OS (145 vs 39 weeks; P&amp;lt;0.001). In four out of thirteen NSCLC patients treated with targeted therapy, resistance mutations were identified using UltraSEEK® at disease progression, implicating the requirement of a therapy switch. Conclusion: Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect both mutations at diagnosis and disease progression, and to monitor treatment response. Citation Format: Paul van der Leest, Melanie Janning, Naomi Rifaela, Maria L. Aguirre Azpurua, Jolanthe Kropidlowski, Sonja Loges, Nicholas Lozano, Alexander Sartori, Darryl Irwin, Pierre-Jean Lamy, T. Jeroen N. Hiltermann, Harry J. Groen, Klaus Pantel, Léon C. van Kempen, Harriet Wikman, Ed Schuuring. Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3411.

Abstract 2144: Holistic sampling of clonal dynamics using cfDNA in lung TRACERx

Abstract Background: Minimal residual disease (MRD) detection using liquid biopsy has the potential to improve patient outcomes in non-small cell lung cancer (NSCLC). Liquid biopsy may also provide representative clonal sampling through the disease course, but current clonal deconvolution methods are ineffective in plasma samples with &amp;lt;1% tumor content, common in the localised or MRD setting. Methods: We analysed 1071 plasma samples from 198 TNM I-III NSCLC patients in TRACERx who underwent multiregion exome sequencing of primary tumor and relapse tissue, with 416 standard of care surveillance scans. Seventy-four patients suffered a relapse. We targeted a median of 200 tumor-specific mutations per patient consisting of clonal and subclonal variants and sequenced cell-free DNA (cfDNA) using anchored multiplex PCR to a median unique depth of 2230X with 5 supporting duplicates. We used library-specific trinucleotide background models to call MRD. We developed ECLIPSE (Extraction of CLonality from LIquid bioPSiEs), to perform formal clonal deconvolution in &amp;lt;1% purity plasma samples by leveraging copy number and mutation clone identities from tumor tissue. Results: Median MRD lead time was 119 days (range 0-1137) in patients with pre-operative circulating tumor DNA (ctDNA) detection. In the 13% of relapse patients lacking pre-operative ctDNA detection the median MRD lead time was 0 days (range 0-589). MRD lead times positively correlated with clonal ctDNA fraction doubling times (DTs). Cancer cell fractions (CCFs) of subclones estimated in plasma with ECLIPSE and tissue collected concurrently were proportional (P &amp;lt; 0.001, R2 = 0.6). Subclonal mutations that would appear clonal in single biopsies could be separated from true clonal mutations using their CCF in plasma (P &amp;lt; 0.001, OR = 0.44), distinguishing clonal from subclonal mutations, where the latter are unlikely to represent effective therapeutic targets. Where cfDNA and tissue was available at relapse we detected 28/29 metastatic tissue subclones in cfDNA with an additional 8 cfDNA-unique subclones. These subclones were more frequently estimated as present in only a subset of metastatic cells using cfDNA (P = 0.008, OR = 5.5) consistent with localisation to unsampled metastatic sites. Metastatic competent subclones had higher CCFs in pre-operative plasma (P &amp;lt; 0.001, OR = 4.5). Shifts in clonal dynamics were concurrent with treatment. Finally, patients with cfDNA-detected polyphyletic metastatic seeding had shorter disease-free survival than those with monophyletic seeding (HR = 2.89, 95% CIs 1.46-5.73). Conclusions: Tumor-informed anchored multiplex PCR most commonly detected MRD before clinical relapse and allowed determination of clonal ctDNA DT. Using ECLIPSE, plasma samples of &amp;lt;1% purity allow formal measurements of clonal dynamics from diagnosis to relapse, which impacts patient outcome and has the potential to guide personalised medicine. Citation Format: Alexander Mark Frankell, Christopher Abbosh, Aaron Garnett, Judit Kisistok, Thomas Harrison, Morgan Weichert, Abel Licon, Selvaraju Veeriah, Bob Daber, Mike Moreau, Aamir Shahpurwalla, Aaron Odell, Adrian Chesh, Kevin Litchfield, Emilia Lim, Daniel E. Cook, Clare Puttick, Maise Al-Bakir, Fabio Gomes, Akshay Patel, Lizi Manzano, Paula Roberts, Ariana Huebner, Nicolas Carey, Joan Riley, Todd Druley, Jacqui A. Shaw, Nicholas McGranahan, Mariam Jamal-Hanjani, Josh Stahl, Nicolai Birkbak, the Lung TRACERx consortium, Charles Swanton. Holistic sampling of clonal dynamics using cfDNA in lung TRACERx [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 2144.

Thwarting of Lphn3 Functions in Cell Motility and Signaling by Cancer-Related GAIN Domain Somatic Mutations.

Cancer progression relies on cellular transition states accompanied by changes in the functionality of adhesion molecules. The gene for adhesion G protein-coupled receptor latrophilin-3 (aGPCR Lphn3 or ADGRL3) is targeted by tumor-specific somatic mutations predominantly affecting the conserved GAIN domain where most aGPCRs are cleaved. However, it is unclear how these GAIN domain-altering mutations impact Lphn3 function. Here, we studied Lphn3 cancer-related mutations as a proxy for revealing unknown GAIN domain functions. We found that while intra-GAIN cleavage efficiency was unaltered, most mutations produced a ligand-specific impairment of Lphn3 intercellular adhesion profile paralleled by an increase in cell-matrix actin-dependent contact structures for cells expressing the select S810L mutation. Aberrant remodeling of the intermediate filament vimentin, which was found to coincide with Lphn3-induced modification of nuclear morphology, had less impact on the nuclei of S810L expressing cells. Notoriously, receptor signaling through G13 protein was deficient for all variants bearing non-homologous amino acid substitutions, including the S810L variant. Analysis of cell migration paradigms revealed a non-cell-autonomous impairment in collective cell migration indistinctly of Lphn3 or its cancer-related variants expression, while cell-autonomous motility was potentiated in the presence of Lphn3, but this effect was abolished in S810L GAIN mutant-expressing cells. These data identify the GAIN domain as an important regulator of Lphn3-dependent cell motility, thus furthering our understanding of cellular and molecular events linking Lphn3 genetic somatic mutations to cancer-relevant pathogenesis mechanisms.

Personalized neoantigen DNA vaccines expand tumor-specific T cells in the periphery which infiltrate the tumor in hepatocellular carcinoma.

2638 Background: Tumor neoantigens are epitopes derived from tumor-specific mutations that can be incorporated in personalized vaccines to prime T cell responses. DNA vaccines delivered with electroporation have recently shown strong CD8 and CD4 T cell responses in clinical trials. In preclinical studies, DNA-encoded neoantigen vaccines have shown induction of CD8 T cells against 50% of predicted high affinity epitopes with the ability to impact tumor growth. Methods: Paired blood and tumor biopsy samples were collected from a patient with hepatocellular carcinoma before and after treatment with GNOS-PV02 (DNA neoantigen targeted vaccine) + plasmid IL-12 + pembrolizumab. Treatment resulted in a partial response with a decrease in tumor size of 44% by RECIST (168 mm to 94 mm). TCRbeta sequencing was performed on all 4 samples and single cell TCR and transcriptome sequencing was performed from T cells isolated from the post-treatment blood sample. Newly identified TCRs in blood and tumor after vaccination were inserted into an expression vector and used to generate engineered TCR T cells. Engineered TCR T cells were tested against the neoantigens included in the vaccine and their responses characterized by flow cytometry. Results: We identified 67,893 new clones in PBMC after vaccination, 3 of which comprised between 0.1 to 1% of the total T cell clones. Moreover, we identified 5126 new clones in the tumor post vaccination, out of these, 3878 (75.68%) were not found within the patient’s pre vaccination PBMCs and 556 (10.86%) were identified within the pre vaccination PBMC pool. Importantly, of the newly identified T cells infiltrating the tumor post vaccination, we observed high frequency TCR clones of which 44 and 7 clones were above 0.1% and 1%, respectively. The majority of the newly identified T cell clones were CD8 T cells (68.75%) with an activated phenotype. Importantly, the 6 most expanded clones in blood were identified to be activated CD8+CD69+ T cells (81.82%). Engineered TCR T cells generated encoding the TCRs of these newly identified CD8 T cells showed activation when exposed to the tumor neoantigens encoded in the neoantigen DNA vaccine GNOS-PV02. Conclusions: GNOS-PV02, a neoantigen DNA vaccine, in combination with plasmid IL-12 and pembrolizumab resulted in expansion of newly identified T cells, primarily activated CD8, which trafficked to the tumor. These new tumor infiltrating T cells showed TCR specificity against tumor neoantigens encoded in GNOS-PV02 and may account for the observed objective decrease in tumor size.

Increased adherence to molecular profiling recommendations in non–small cell lung cancer through use of prior authorization and peer review.

9129 Background: National oncology guidelines advocate for broad molecular profiling to detect potentially actionable driver mutations prior to initiation of first-line therapy for patients with advanced non-small cell lung cancer (NSCLC). Results of genomic tests can confirm the presence of specific cancer tumor mutations that are best treated with more targeted therapies. Recent studies have shown that up to 30% of lung cancer patients may not receive the most effective treatment due to a lack of guideline-concordant genomic testing. We analyzed data from our prior authorization medical oncology program to determine adherence to national guidelines recommendations, and then developed a program to improve adherence to broad molecular profiling utilizing our proprietary prior authorization algorithms and our proactive peer consultation model. Methods: eviCore healthcare uses the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) to support its proprietary program for medical oncology drug management, and requires attestation on the results of NCCN recommended genomic/molecular test results in advanced NSCLC during prior authorization of systemic therapy. In November 2019, we implemented a next-generation sequencing (NGS) testing results requirement for metastatic non-squamous NSCLC patients prior to first-line therapy approval. If testing had not been completed, a peer to peer consultation was performed to advocate for testing prior to initiation of chemoimmunotherapy regimens. The rate of appropriate tumor testing is defined by provider attestation of test results prior to initiation of therapy. We define the control period (pre- implementation) as October 2018 through September 2019 ( n = 667). The measurement period (post-implementation) is January 2020 through December 2020 ( n = 390). Results: From pre to post there was a 35% increase in testing adherence (p &lt; 0.001). After the testing requirement was implemented, the appropriate tumor testing rate increased to 99% by the second half of 2020, compared to the control period (62%). Additionally, after the testing requirement began, pembrolizumab utilization for advanced stage NSCLC decreased by a net 4.2% per month. This decrease in pembrolizumab utilization likely reflected patients being matched to more appropriate targeted therapy based on genomic testing results. Conclusions: We demonstrate that by combining medical oncology prior authorization with focused peer consultation, adherence to national society recommendations for broad molecular profiling prior to initiating first line therapy in advanced NSCLC can be significantly improved. By improving testing adherence, the most effective and appropriate therapy can be matched to the patient.

P-174 OPTIMISE: Optimization of treatment selection and follow-up in oligometastatic colorectal cancer – a ctDNA-guided phase II randomized approach with a run-in feasibility part

Patients with detectable circulating tumor DNA (ctDNA) after surgery for colorectal cancer are considered to have microscopic residual disease and consequently a high risk of recurrence. The same applies to patients after local ablative treatment (LAT) for metastatic colorectal cancer (mCRC). We observed a temporal clearance of ctDNA during adjuvant chemotherapy, but ctDNA recurrence after therapy termination. This indicate that patients with ctDNA positive status may need more intensified adjuvant chemotherapy than standard of care (SOC). In OPTIMISE we investigate the use of ctDNA for treatment decisions on adjuvant chemotherapy after LAT for mCRC. We hypothesis that recurrence can be prevented with intensified adjuvant chemotherapy in patients with ctDNA positive blood samples post ablations, and that patients without detectable ctDNA have undergone successful radical treatment and will not need adjuvant chemotherapy. OPTIMISE is an open label 1:1 randomized multicentre, phase II study, comparing ctDNA guided adjuvant chemotherapy against standard of care (SOC) approach, with a run-in phase investigating feasibility measures. Eligibility criteria comprise, Radical intended treatment for metastatic spread from CRC (resection, radiofrequency ablation, stereotactic body radiation therapy, or other experimental local treatment), No evidence of further disease according to SOC, Age ≥ 18 years, ECOG performance status 0-2, Clinically eligible for adjuvant triple chemotherapy at investigators decision, Adequate bone marrow function allowing systemic chemotherapy, Anticonception for fertile women and for the male patients with a fertile partner, Written and verbally informed consent. Exclusion criteria are, Neuropathy CTCAE grade > 1, Other malignant tumour within 5 year except non-melanoma skin cancer or carcinoma in situ cervicis uteri, Pregnant or breastfeeding women, Intolerance or allergy to 5-FU, leucovorin, oxaliplatin, irinotecan or capecitabin Before randomization participants undergo a PET-CT scan to exclude metabolically detectable lesions. In ARM A patients are treated according to SOC, and blood samples collected for later retrospective ctDNA analysis. In ARM B, patients follow a ctDNA-guided strategy; ctDNA positivity lead to escalation strategy with 4 months of FOLFOXIRI followed by 2 months of 5-FU, ctDNA negative status to de-escalation strategy by shared decision-making. Endpoints Primary endpoint; fraction of patients alive without recurrence 2 years after inclusion. Secondary endpoints; CTCAE grade 3-5 toxicity, ctDNA negativity (6 months + 1 year), Time to molecular -biological and radiological recurrence, Rate of local and distant relapse, Overall survival, QoL and Cost-effectiveness. Laboratory methods. Blood samples are collected in streck-tubes and analysed for tumor specific mutations and methylations by multiplex ddPCRs in two Danish laboratories (with tumor-agnostic approach). Sample size is 350 but will be revised based on findings in the run-in phase. Feasibility measures for the run-in phase are: Inclusion of 30 patients over 12 months in two Danish Hospitals, Compliance with randomization > 80%, Rate of unexpected PET-CT positive cases 20%, Eligibility for triple chemotherapy > 80%. The study is including. NCT04680260. Sponsors.

Roles of Exosome Genomic DNA in Colorectal Cancer.

Exosomes are extracellular vesicles that mediate cell-to-cell communication. Bioactive substances such as DNA, RNA, lipids, and proteins are present in it, and they play an essential role in the pathogenesis of colorectal cancer (CRC). The role of RNA and protein in exosomes has been extensively studied. Exosome DNA has recently attracted the attention of a great deal of scientists. According to studies, exosome DNA mainly contains genomic DNA (gDNA) and mitochondrial DNA (mtDNA), of which exosome gDNA is widely used in liquid biopsy of CRC. It includes a variety of clinically relevant tumor-specific mutation genes. In addition to liquid biopsy, researchers find that exosome gDNA regulates immune and metabolic functions in CRC, making it an important research object. However, the primary research on exosome gDNA is still limited. Here, we describe the occurrence and composition of exosomes. Summarize the essential characteristics and mode of action of exosome gDNA. Remarkably, this paper constitutes a comprehensive summary on the role of exosome gDNA on CRC with the intent of providing a theoretical basis and reference for early diagnosis and clinical treatment of cancer.

Abstract PR005: TP53 field defects in uterine fluid are associated with ovarian cancer risk

Abstract Background: High grade serous ovarian cancer (HGSC), the most common and aggressive type of ovarian cancer, is nearly always driven by TP53 mutations, which can be detected in uterine lavages using ultra-deep duplex sequencing. While this finding supports the development of a uterine lavage assay for early HGSC detection, age-related TP53 mutations compromise specificity. The goal of this study was to thoroughly characterize the nature of TP53 mutations identified in uterine lavage of women with and without HGSC to better understand TP53 clonal evolution during aging and to determine its association to HGSC. Methods: Uterine lavages were collected preoperatively in 54 “average-risk” women undergoing gynecological surgery for suspicious masses (34 women ultimately had HGSC, 20 were cancer free) and in 98 “high-risk” women undergoing risk-reducing salpingo-oopherectomy (17 were found to have HGSC or serous tubal intraepithelial carcinomas (STICs), 81 were cancer-free). Ultra-deep (&amp;gt;10,000x), ultra-accurate sequencing of TP53 was performed on uterine lavage DNA using duplex sequencing. Tumors and STICs were also duplex sequenced to determine TP53 tumor-driving mutations. The frequency of TP53 mutations and large TP53 mutant clones (those found in &amp;gt;2 duplex reads) was compared between women with and without HGSC. Results: The tumor-specific TP53 mutation was detected in 53% of lavages from average-risk women with HGSC and in 22% of lavages from high-risk women with HGSC/STICs. As in prior studies, TP53 biological background mutations were identified in nearly all women and their frequency was age-related. TP53 background mutations, however, were more abundant in women with prior chemotherapy, regardless of their cancer status or age. In women without prior chemotherapy and without high-risk of HGSC, having HGSC was associated with a significantly higher frequency of large TP53 mutant clones (p=0.007) and large TP53 mutant clones containing pathogenic mutations (p=0.007) or TP53 mutations commonly found in cancer (p=0.001). Interestingly, women at high risk of HGSC also carried high frequency of large and pathogenic TP53 mutant clones in lavage, even in those cases in which cancer or STICs were not identified. Conclusions: Ultra-sensitive duplex sequencing enabled detection of a patient’s HGSC or STIC driver mutations, respectively, in about half and a quarter of uterine lavages. However, of even greater interest, we observed a significant excess of large and pathogenic TP53 mutant clones in lavages of women with HGSC that were not present in the tumor. These results indicate that TP53 somatic evolution is associated with HGSC development and suggest that the measurement of TP53 mutant clones in uterine lavages harbors value as a predictor of ovarian cancer risk. Citation Format: Rosana Risques, Thomas H. Smith, Zachary K. Norgaard, Roniz Katz, Fang Yin Lo, Elizabeth K. Schmidt, Jacob E. Higgins, Martin Filipits, Intidhar Labidi-Galy, David Cibula, Lukáš Dostálek, Gabriel Jelenek, Magdalena Plch, Jiří Bouda, Alexander Mustea, Mateja Condic, Sabine Grill, Noreen Gleeson, Peter Oppelt, Gunda Pristauz-Telsnigg, Adriaan Vanderstichele, Siel Obrecht, Adam Rosenthal, Paul Speiser, Jesse Salk. TP53 field defects in uterine fluid are associated with ovarian cancer risk [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR005.

Diagnostic potential of extracellular vesicles in meningioma patients.

Extracellular vesicles (EVs) play an important role in cell-cell communication, and tumor-derived EVs circulating in patient blood can serve as biomarkers. Here, we investigated the potential role of plasma EVs in meningioma patients for tumor detection and determined whether EVs secreted by meningioma cells reflect epigenetic, genomic, and proteomic alterations of original tumors. EV concentrations were quantified in patient plasma (n = 46). Short-term meningioma cultures were established (n = 26) and secreted EVs were isolated. Methylation and copy number profiling was performed using 850k arrays, and mutations were identified by targeted gene panel sequencing. Differential quantitative mass spectrometry was employed for proteomic analysis. Levels of circulating EVs were elevated in meningioma patients compared to healthy individuals, and the plasma EV concentration correlated with malignancy grade and extent of peritumoral edema. Postoperatively, EV counts dropped to normal levels, and the magnitude of the postoperative decrease was associated with extent of tumor resection. Methylation profiling of EV-DNA allowed correct tumor classification as meningioma in all investigated cases, and accurate methylation subclass assignment in almost all cases. Copy number variations present in tumors, as well as tumor-specific mutations were faithfully reflected in meningioma EV-DNA. Proteomic EV profiling did not permit original tumor identification but revealed tumor-associated proteins that could potentially be utilized to enrich meningioma EVs from biofluids. Elevated EV levels in meningioma patient plasma could aid in tumor diagnosis and assessment of treatment response. Meningioma EV-DNA mirrors genetic and epigenetic tumor alterations and facilitates molecular tumor classification.

PD58-03 DETECTION OF INDIVIDUALIZED MUTATION USING PLASMA AND URINARY CIRCULATING TUMOR DNA IN PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA

You have accessJournal of UrologyCME1 May 2022PD58-03 DETECTION OF INDIVIDUALIZED MUTATION USING PLASMA AND URINARY CIRCULATING TUMOR DNA IN PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA Daichi Tamura, Masakazu Abe, Renpei Kato, Yoichiro Kato, Shigekatsu Maekawa, Mitsugu Kanehira, Ryo Takata, Yutaka Suzuki, Hidewaki Nakagawa, Satoshi Nishizuka, and Wataru Obara Daichi TamuraDaichi Tamura More articles by this author , Masakazu AbeMasakazu Abe More articles by this author , Renpei KatoRenpei Kato More articles by this author , Yoichiro KatoYoichiro Kato More articles by this author , Shigekatsu MaekawaShigekatsu Maekawa More articles by this author , Mitsugu KanehiraMitsugu Kanehira More articles by this author , Ryo TakataRyo Takata More articles by this author , Yutaka SuzukiYutaka Suzuki More articles by this author , Hidewaki NakagawaHidewaki Nakagawa More articles by this author , Satoshi NishizukaSatoshi Nishizuka More articles by this author , and Wataru ObaraWataru Obara More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002643.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper tract urothelial carcinoma (UTUC) is an aggressive disease which have high recurrence after surgery. Current standard methods for the diagnosis and detection of recurrence are invasive or low sensitivity. Circulating tumor DNA (ctDNA) analysis using digital polymerase chain reaction (dPCR) has demonstrated promising results for monitoring as recurrence in several cancers. The aim of this study was to evaluate the validity of plasma and urinary ctDNA as a tumor biomarker for UTUC among a perioperative period. METHODS: This study included 23 patients who underwent radical nephroureterectomy (RNU) diagnosed as UTUC from January 2019 to December 2020. In each patient, whole exome sequencing (WES) of tumor DNA and corresponding peripheral blood mononuclear cell (PBMC) DNA were performed by next generation sequencer. Tumor specific mutations were identified by comparing the single nucleotide variants from tumors and PBMCs. To investigate ctDNA by dPCR analysis, we designed primers and probes for each mutation. We also obtained plasma and urine ctDNA from each patient. Longitudinal variant allele frequency (VAF) of ctDNA were plot on a perioperative period. RESULTS: The median age was 71 years old (range 57-82). 13 cases were renal pelvis cancer, and 10 cases were ureteral cancer. The median tumor mutation burden in all cases was 3.02/Mb (range 0.00-15.97). Tumor specific mutations such as TERT, PIK3CA and p53 gene were selected by individualized patient, and performed plasma and urinary ctDNA monitoring by dPCR in 7 patients. Tumor specific mutations were detected in preoperative samples and VAFs of ctDNA decreased quickly after RNU in 5 patients. Tumor specific mutations were not detected in 2 patients who were pT0 due to neoadjuvant chemotherapy. CONCLUSIONS: VAFs in plasma and urinary ctDNA were associated with tumor burden. Individualized ctDNA monitoring could be a useful biomarker for UTUC. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e1012 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daichi Tamura More articles by this author Masakazu Abe More articles by this author Renpei Kato More articles by this author Yoichiro Kato More articles by this author Shigekatsu Maekawa More articles by this author Mitsugu Kanehira More articles by this author Ryo Takata More articles by this author Yutaka Suzuki More articles by this author Hidewaki Nakagawa More articles by this author Satoshi Nishizuka More articles by this author Wataru Obara More articles by this author Expand All Advertisement PDF DownloadLoading ...