bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 456: Liquid biopsy for patient stratification and monitoring of dacomitinib clinical trial in patients with EGFR amplified recurrent glioblastoma
Authors: Yekula Anudeep , Kitchen Robert R. , Chakrabortty Sudipto K. , Carter Bob S. , Skog Johan , Balaj Leonora
Published: July 2021

Introduction

Liquid biopsy for the detection and monitoring of brain tumors is of significant clinical interest. This ability to non-invasively profile tumors, opens new avenues for diagnosis, patient stratification to novel therapies, as well as monitoring. Here, we provide evidence of extracellular vesicle EV RNA-based diagnosis, patient stratification, and assessment of response to therapy in the setting of a clinical trial evaluating the efficacy of dacomitinib, EGFR tyrosine kinase inhibitor in patients with recurrent, EGFR amplified GBM in a multi-armed, multi-institutional clinical trial (NCT01112527).

Methods

We performed RNASeq on long RNA extracted from the serum samples, pre-treatment and 1-month post-treatment.

Results

Firstly, our novel RNASeq workflow on EV derived long RNA allowed the detection of thousands of protein coding genes, lincRNAs and antisense RNAs enabling the study of a wider repertoire of potential RNA based biomarkers of GBM.

Secondly, we observed a differential expression profile is serum EV RNA of GBM patients and healthy controls. Combining our findings with TCGA data and literature screening, we generated a 25 gene signature representative of critical pathways in several hallmarks of cancer including gliogenesis, epithelial to mesenchymal transition, invasion, regulation of stemness, apoptosis and cell cycle regulation, immune regulation and metabolic remodeling.

Thirdly, we observed a differential expression profile is serum EV RNA of responders compared to non-responders in pre-treatment serum. Specifically, the EV mRNAs ZNF35 and LAMTOR2 distinguish responders from non-responders (p-adjusted = 2.6E-8 and 2.4E-6, respectively) allowing potential patient stratification.

Finally, we also observed a differential expression profile is serum EV RNA of responders compared to non-responders in post-treatment serum. EV mRNA DNMT3A is significantly enriched (p-adjusted = 1.8E-4) in post-treatment serum of responders compared to non-responders to dacomitinib allowing potential monitoring of response to therapy.

Conclusion

This study is unique because it represents the first longitudinal profiling of the EV RNA in a cohort of genomically selected GBM patients. These findings are a tantalizing step toward liquid biopsy-based biomarkers for the detection of GBM, as well as patient stratification and monitoring.

Citation Format: Anudeep Yekula, Robert R. Kitchen, Sudipto K. Chakrabortty, Bob S. Carter, Johan Skog, Leonora Balaj. Liquid biopsy for patient stratification and monitoring of dacomitinib clinical trial in patients with EGFR amplified recurrent glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 456.
Abstract 467: Detecting ALK rearrangement by RT-PCR: A superior approach to next generation sequencing
Authors: Kuang Yukun , Xu Peihang , Wang Jiyu , Zheng Yifan , Sun Xue , Li Zimu , Gan RunJing , Li Huixia , Guo Yubiao , Zhu Guanshan , Zhu Changbin , Ke Zunfu , Tang Kejing
Published: July 2021

Background: Precisely detecting anaplastic lymphoma kinase (ALK) rearrangement ensures ideal efficacy of ALK targeted tyrosine kinase inhibitors (ALK-TKIs). Next generation sequencing (NGS) has been widely used in clinics, while, numbers of studies indicated NGS would yield false negative outcome, especially for fusion analysis. Instead, more fusions can be detected by RT-PCR at transcription level. This study aims to compare the capacity of RT-PCR and NGS approach to detect ALK rearrangement in Chinese non-small-cell lung cancer (NSCLC) patients.

Methods: Formalin-fixed paraffin-embedded (FFPE) tissues from 153 patients pathologically diagnosed as NSCLC with eligible ALK status given by previous NGS testing (29 patients were ALK positive and 124 patients were ALK negative) were collected from November 2017 to October 2019. RT-PCR were applied to detect ALK rearrangements in both groups. For samples with discordant result of ALK status, fluorescence in situ hybridization (FISH) or Sanger's sequencing were used for validation.

Results: One hundred and twenty-four samples (26 of 30 ALK positive samples; 98 of 124 ALK negative samples) were successfully subjected to RT-PCR. Five of 98 ALK negative samples given by NGS test were re-defined as ALK positive by RT-PCR. Meanwhile, in 26 ALK positive samples, RT-

PCR yielded acceptable result with 96.15% of concordance rate. Only one NGS-defined ALK positive sample was uncovered by RT-PCR due to a rare ALK rearrangement. Four of 6 RT-PCR defined ALK positive cases (3 RT-PCR positive and 1 NGS positive) were validated by either FISH or sanger's sequencing, which resulted positive result.

Conclusions: RT-PCR displays reliable and superior capacity to detect ALK fusion in NSCLC patients.

Citation Format: Yukun Kuang, Peihang Xu, Jiyu Wang, Yifan Zheng, Xue Sun, Zimu Li, RunJing Gan, Huixia Li, Yubiao Guo, Guanshan Zhu, Changbin Zhu, Zunfu Ke, Kejing Tang. Detecting ALK rearrangement by RT-PCR: A superior approach to next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 467.
Abstract 475: Improving liposarcoma diagnosis through cross-flow microfiltration
Authors: Casadei Lucia , Sarchet Patricia , Choudhury Adarsh , Prakash Shaurya , Pollock Raphael
Published: July 2021

Introduction: Dedifferentiated liposarcoma (DDLPS) is frequently diagnosed late and patients typically respondpoorly to treatments bringing to an overall survival rate of only 10% at 10 years. Prognosis isenhanced by early detection of recurrent lesions, but current diagnostic methods rely on invasivetissue biopsies combined with whole or part-body imaging. These diagnostic and prognosticchallenges prompted us to explore the use of circulating biomolecules as potential biomarkersaccessible through bodily fluids. Extracellular Vesicles (EVs) are membrane vesicles containingproteins and nucleic acids that circulate in the blood, so they are an emerging class of easilyaccessible blood biomarkers. We recently identified extracellular vesicle EV-MDM2 as liquidbiopsy biomarker for disease identification in DDLPS. Unfortunately, due to these challenges forisolation and capture of EVs, despite the potential diagnostic and prognostic utility, the practicalrelevance of using EVs for routine analysis remains limited.

Methods: A microfluidic device was fabricated via standard soft lithography in PDMS(polydimethylsiloxane). A 5 x 5 mm2 nanocapillary array membrane (nominal capillary diameterof 0.2 µm) was sandwiched between two microchannels for a vertically aligned configuration withan injection and separation channel (each 500 µm wide and 150 µm high) perpendicular to eachother. The separation channel surface was functionalized with the anti-CD63 antibody forcapturing EVs.

Results: We present here a facile approach to fabricate and operate a micro-nanofluidic device that usescross-flow filtration to isolate and capture liposarcoma derived EVs. Our methodology integratesthe unit operations of size based separation with immunoaffinity-based capture of extracellularvesicles in the same device. The eluted media collected showed ~84% extracellular vesiclerecovery from the liposarcoma cell conditioned media and ~38% extracellular vesicle recoveryfrom dedifferentiated liposarcoma patient serum when compared to extracellular vesicle isolationand subsequent quantification by ultracentrifugation. The results reported here also show a five-fold increase in amount of critical liposarcoma relevant extracellular vesicle cargo obtained in30 minutes presenting a significant advance over existing state-of-art.

Conclusion: In conclusion we developed a cross-flow filtration micro-nanofluidic device for isolation, selectivecapture and release of liposarcoma EVs.

Citation Format: Lucia Casadei, Patricia Sarchet, Adarsh Choudhury, Shaurya Prakash, Raphael Pollock. Improving liposarcoma diagnosis through cross-flow microfiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 475.
Abstract 49: Brain penetrant HDAC inhibitor RG2833 induces DIPG cell death and synergizes with lomustine
Authors: Barnett Katherine K. , Novak Orlandi , Eberhart Charles , Raabe Eric
Published: July 2021

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor. The pan-histone deacetylase (HDAC) inhibitor panobinostat showed great activity against DIPG in pre-clinical models, but has poor blood brain barrier penetration and demonstrated significant toxicity in clinical trials. RG2833 (RGFP109) is a selective HDAC1/3 inhibitor with established brain penetration. In clinical trials, the Cmax(plasma) of RG2833 was 32uM. RG2833 demonstrated cytotoxicity against temozolomide-resistant glioblastoma and downregulated the NFĸB pathway. Because this pathway is overexpressed in DIPG and may play a role in DIPG cell growth and survival, we hypothesized that RG2833 would kill DIPG cells. Treatment of DIPG cell lines with RG2833 as a single agent inhibits cell growth in the 5 to 10μM range (MTS assay for HSJD007 p=0.0004 10μM vs DMSO, JHH-DIPG1 p=0.001 10μM vs DMSO, SF-7761 p=0.04 10μM vs DMSO, SU-DIPG13 p=0.01 10μM vs DMSO by t-test). RG2833 induces apoptosis by 48 hours as measured by Western blot for cPARP and cleaved caspase 3 immunofluorescence (HSJD007 p<0.003 8μM vs DMSO, JHH-DIPG1 p=0.0026 10μM vs DMSO by t-test). RG2833 also slows cell proliferation as measured by Western blot for phospho-Rb and immunofluorescence for BrdU (HSJD007 p=0.008 8μM vs DMSO, JHH-DIPG1 p=0.0002 10μM vs DMSO by t-test). Western blot confirmed a dose-dependent increase in histone 3 acetylation with RG2833 treatment. We detected increased acetylated p65 and decreased expression of the NFĸB regulated pro-survival genes BCL2, BCL-xL, and XIAP with RG2833 treatment in vitro. Treatment of DIPG flank tumor mouse models with RG2833 alone for 5 days suppressed tumor growth (p< 0.005 by t-test). Combination treatment with RG2833 and lomustine was synergistic against DIPG cell lines in vitro (ZIP synergy scores by SynergyFinder for JHH-DIPG1 17.8 and HSJD007 17.7). We will next assess combination treatment in vitro and in vivo for apoptotic effects. This data indicates that selective HDAC inhibitor RG2833 may be a promising therapeutic candidate for DIPG.

Citation Format: Katherine K. Barnett, Orlandi Novak, Charles Eberhart, Eric Raabe. Brain penetrant HDAC inhibitor RG2833 induces DIPG cell death and synergizes with lomustine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 49.
Abstract 495: TNFR2 and PD-1/PD-L1/TNFR2 humanized mice aiding exploration of combination strategies to overcome tumor immune escape mechanisms
Authors: Li Huilin , Zhou Xiaofei , Zhang Jing , Chromikova Veronika , Lin Qingcong
Published: July 2021

Tumor necrosis factor receptor 2 (TNFR2), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), is a transmembrane protein, which can mediate both pro- and anti-inflammatory activities of T cells. TNFR2 is mainly expressed on the surface of activated effector T cells and regulatory T cells (Tregs) and promotes proliferation and survival of Tregs through nuclear factor kappa B (NF-κB). Blockade of TNF-TNFR2 interaction with monoclonal antibodies can inhibit the activation of Tregs, possibly resulting in inhibition of their function and/or reduction of their numbers. Yet, until recently the potential of TNFR2 as a therapeutic target for cancer therapy has been underappreciated. Discovery of anti-TNFR2 antibodies, developed to inhibit NF-κB driven growth, has revived excitement for the use of TNFR2as a cancer therapy target and stressed the demand for suitable pre-clinical models to evaluate the safety and efficacy of TNFR2-targeted therapeutics. Biocytogen has generated a humanized TNFR2 mouse model for both in vitro function validation of signaling pathways and in vivo efficacy evaluation of TNFR2 antibodies. In this model, the exons 2~6 of mouse Tnfrsf1b gene which encode the extracellular domain were replaced by human TNFRSF1B counterparts. In the humanized mouse, human TNFR2 was detectable on Tregs in the spleen and the anti-human TNFR2 antibodies associated well with the splenocytes. In addition, anti-human TNFR2 antibodies bound well to CD3+ T cells and inhibited tumor growth. Furthermore, Biocytogen has also generated PD-1/PD-L1/TNFR2 multi-gene humanized mice, in which blood cell composition, morphology and the levels of ALT, AST and other indicators were similar to the wild-type counterpart. Basal leukocyte subpopulations including T/B/NK cells, DC, granulocytes, and monocytes/macrophages, were similar between humanized and wild-type mice. Moreover, using the MC38 tumor model, the combination of anti-TNFR2 antibody and anti-PD-1 (or anti-PD-L1) shows enhanced effect compared to the individual groups, demonstrating that the PD-1/PD-L1/TNFR2 humanized mice have a potential to provide a powerful preclinical model for in vivo evaluation of combination therapies. In conclusion, TNFR2 and PD-1/PD-L1/TNFR2 humanized mice are a useful tool to explore anti-PD-1, anti-PD-L1 and anti-TNFR2 combination therapy strategies with promising activity and low cytotoxicity.

Citation Format: Huilin Li, Xiaofei Zhou, Jing Zhang, Veronika Chromikova, Qingcong Lin. TNFR2 and PD-1/PD-L1/TNFR2 humanized mice aiding exploration of combination strategies to overcome tumor immune escape mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 495.
Abstract 511: Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing
Authors: Toro Michelle , Lowman Geoffrey , Pickle Loni , Ostresh Stephanie , Andersen Mark , Sarda Shrutii , Yang Chenchen
Published: July 2021

Background

Traditional next generation sequencing methods for quantifying somatic hypermutation (SHM) rely on multiplex primers targeting either the framework 1 (FR1) or Leader regions of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA templates. Ion AmpliSeq primer panels for SHM evaluation were compared using both DNA and RNA input. Performance was compared using SHM values obtained from RNA samples amplified using FR1 variable gene primers in combination with constant gene primers to determine each IGH isotype and subtype in a single PCR reaction. Comparison of SHM frequencies measured from matched RNA and DNA samples were used to determine the feasibility for use of RNA in the study of SHM as well as comparison of the performance of Leader and FR1 gene primers in DNA studies.

Methods

Two multiplex primer panels were developed for DNA templates. These panels target the Leader or FR1 regions of the IGHV gene and the IGHJ gene. RNA samples were surveyed using FR1 and constant region primers. Comparisons of SHM frequency using 200ng of gDNA and 25ng of RNA from B cell lines and clinical research samples. Sequencing was accomplished using the Ion Gene Studio S5, while clonotyping, isotype determination, and somatic hypermutation analysis was performed using Ion Reporter 5.16 analysis software.

Results

Both RNA and DNA input assay workflows were able to correctly determine the SHM status of all rearrangements tested. IGHV SHM values were highly concordant between both RNA and DNA approaches. Additionally, SHM values derived from FR1 targeting variable gene primers delivered concordant results compared to Leader targeting variable gene primers when using DNA input across a wide range of SHM frequencies tested.

Conclusions

These results support the ability of highly multiplexed long-read NGS assays to accurately quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader targeting primers using DNA input. RNA based NGS methods benefit from lower sample requirements as well as the addition of isotype (and subtype) identification, opening new research areas for study of the B cell immune repertoire.

Citation Format: Michelle Toro, Geoffrey Lowman, Loni Pickle, Stephanie Ostresh, Mark Andersen, Shrutii Sarda, Chenchen Yang. Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 511.
Abstract 541: Liquid biopsy for molecular characterization and monitoring in patients with metastatic colorectal cancer
Authors: Urbini Milena , Marisi Giorgia , Passardi Alessandro , Molinari Chiara , Canale Matteo , Matteucci Laura , Tedaldi Gianluca , Martinelli Giovanni , Frassineti Giovanni L. , Paola Ulivi
Published: July 2021

The use of liquid biopsy in the clinical management of colorectal cancer (CRC) patients is not yet a routine clinical practice, although it could give important information for tumor baseline characterization and for monitoring during treatment. In this retrospective exploratory study, we examined plasma-circulating tumor DNA (ctDNA) in a series of 27 metastatic CRC patients receiving chemotherapy plus bevacizumab as firstline treatment, both at baseline and at progression. All patients were characterized for RAS and BRAF status on tumor tissue before the start of treatment: 70% of patients were RAS-mutated(16 KRAS, 3 NRAS),15% were BRAF mutated and 15% were RAS/BRAF wild-type. Detection of ctDNA mutations were performed using Oncomine™Colon cfDNA Assay on Ion GeneStudio™ S5 System (Thermofisher). All mutations observed in the tissue were identified also in the baseline ctDNA, excluding one BRAF mutated case that was not detected in the ctDNA. Additional mutations on TP53, APC, PIK3CA, FBXW7 were detected respectively in 11, 12, 3 and 2 cases. TP53 mutations were identified also in 3 RAS/BRAF wild-type cases. Baseline RAS/BRAF variant allele frequency (VAF) lower than 10% was found associated with longer progression free survival (PFS) (log-rank p-value=0.027) (Table 1). At progression, 80% of patients carried the same mutations identified at baseline, and 7 cases carried additional mutations of TP53. Predominantly mutations has a lower VAF at progression with respect to baseline, however RAS/BRAF VAF at progression was found not associated with PFS. In conclusion, we report a concordance-rate >96% between ctDNA and tissue demonstrating the high sensibility of liquid biopsy for detection of mutations in metastatic CRC. Thus, ctDNA could be useful for monitoring of minimal residual disease and tumor heterogeneity during therapeutic treatment. These data, if confirmed in a larger cohort, might support the use of liquid biopsy in the clinical management of CRC patients.

Table 1.Relationships between RAS/BRAF mutation VAF and progression free survival during treatmentTimeConditionN. of casesMedian PFS (range), daysχ2p-valueBaselineVAF<10%11504 (244-843)4,8960,0269VAF>10%11373 (41-719)ProgressionVAF<10%14414 (110-763)0,32390,5693VAF>10%8412 (41-843)

Citation Format: Milena Urbini, Giorgia Marisi, Alessandro Passardi, Chiara Molinari, Matteo Canale, Laura Matteucci, Gianluca Tedaldi, Giovanni Martinelli, Giovanni L. Frassineti, Ulivi Paola. Liquid biopsy for molecular characterization and monitoring in patients with metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 541.
Abstract 545: Detection of liver-derived hepatitis B virus-host junction sequences in urine of hepatitis B infected patients for noninvasive disease monitoring
Authors: Lin Selena , Kao Yu-lan , Su Robin , Chang Ting-Tsung , Lin Yih-Jyh , Cui Yixiao , Hann Hie-Won , Park Grace , Song Wei , Su Ying-Hsiu
Published: July 2021

Integrated hepatitis B virus (HBV) DNA detected in more than 85% of HBV-infected hepatocellular carcinoma (HBV-HCC) can cause insertional mutagenesis, chromosomal instability, sustained viral protein expression, and/or immune-mediated inflammation leading to HCC carcinogenesis. An opportunistic outcome of HBV DNA integration events, which occur randomly into the host genome, is the creation of a unique HBV-host junction sequence (HBV-JS) in individually infected hepatocytes representing a specific molecular signature of that cell. A noninvasive approach to detect HBV-JS's will enable frequent monitoring and aid in assessing HBV treatment efficacy and HCC disease progression. Here, we assessed the feasibility of detecting HBV-JS's in urine of HBV-infected patients as a proof-of-concept for utilizing urine as a noninvasive HBV-JS liquid biopsy for HBV-related disease monitoring. Utilizing an in-house developed HBV primer extension capture (PEC) NGS assay, we assessed early-stage HBV-patient tumor tissue and matched urine (n=8). Five of 8 urine samples contained detectable HCC tissue-derived HBV-JS's, including a TERT junction sequence. Next, we assessed 32 urine specimens collected from 28 HBV-infected patients including hepatitis (n=5), cirrhosis (n=11), HCC (n=4), and post-HCC (n=8). Interestingly, all urine samples contained HBV-JS sequences with 30 of 32 urines containing integrations in gene-coding regions. Of 351 unique HBV-JS in gene-coding regions identified in urine, 11 HBV-JS's have also been previously reported in tissue of HCC patients, including TERT. All eleven HBV-JS's were identified in cirrhosis or HCC patient cohorts. Furthermore, the distribution of urinary integrated HBV DNA in the HBV genome in all disease categories was found predominantly clustered in HBV DR1-2 (>70%), an integration hotspot, consistent with findings in tissue. Altogether this support the potential of urine as a noninvasive HBV-JS liquid biopsy to monitor HBV-infected patients for disease progression and treatment efficacy.

Citation Format: Selena Lin, Yu-lan Kao, Robin Su, Ting-Tsung Chang, Yih-Jyh Lin, Yixiao Cui, Hie-Won Hann, Grace Park, Wei Song, Ying-Hsiu Su. Detection of liver-derived hepatitis B virus-host junction sequences in urine of hepatitis B infected patients for noninvasive disease monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 545.
Abstract 553: Personalized circulating tumor DNA analysis in head and neck squamous cell carcinoma: Preliminary results of the Liquid BIOpsy for MiNimal RESidual DiSease Detection in Head and NeckSquamous Cell Carcinoma (LIONESS) study
Authors: Flach Susanne , Howarth Karen , Hackinger Sophie , Pipinikas Christodoulos , McLay Kirsten , Marsico Giovanni , Walz Christoph , Gires Olivier , Canis Martin , Baumeister Philipp
Published: July 2021

Introduction

Head and neck squamous cell carcinoma (HNSCC) remains a substantial burden to global health. Despite evolving therapies, 5-year survival is less than 50% and unlike other cancers, reliable biomarkers to monitor treatment response do not exist. Cell-free circulating tumor DNA (ctDNA) is an emerging biomarker but has not yet been studied sufficiently for HNSCC. The detection of ctDNA as a marker of minimal residual disease following curative-intent treatment holds promise for identifying patients at an increased risk of relapse, who may benefit from adjuvant radio(chemo)therapy or facilitate close monitoring with repeat resection if needed.

Methods

We conducted a single-center prospective experimental evidence-generating cohort study to assess ctDNA in 30 patients with p16-negative HNSCC (stages I-IVB) who received primary surgical treatment with curative intent at our institution. Whole exome sequencing (WES) was performed on formalin-fixed paraffin-embedded tumor tissue to a median depth of 250x. For each patient, we selected up to 48 somatic variants for personalized ctDNA assay design. We used the RaDaRTM assay to analyze serial pre- and post-operative plasma samples (range 2-6) for evidence of minimal residual disease or recurrence.

Results

In a subset of patients analyzed to evaluate the performance of RaDaR, personalized panels were designed with between 34 and 48 somatic variants (median 48). Preliminary data shows 100% ctDNA detection in baseline samples taken prior to surgery at tumor fractions ranging from 312 ppm (equivalent to 0.03% AF) to 7579 ppm (equivalent to 0.76% AF). In post-surgery samples, ctDNA could be detected at levels as low as 26 ppm (equivalent to 0.0026% AF). Analysis of follow-up plasma samples will be presented along with data from the full patient cohort.

Conclusions

This study illustrates the potential of ctDNA as a biomarker in HNSCC and demonstrates the feasibility of personalized ctDNA assays for the detection of minimal residual disease post-treatment and for monitoring for early detection of relapse.

Citation Format: Susanne Flach, Karen Howarth, Sophie Hackinger, Christodoulos Pipinikas, Kirsten McLay, Giovanni Marsico, Christoph Walz, Olivier Gires, Martin Canis, Philipp Baumeister. Personalized circulating tumor DNA analysis in head and neck squamous cell carcinoma: Preliminary results of the Liquid BIOpsy for MiNimal RESidual DiSease Detection in Head and NeckSquamous Cell Carcinoma (LIONESS) study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 553.
Abstract 556: Highly complex DNA sequencing library preparation for cfDNA enrichment panels using a single-stranded approach
Authors: Vaske Charles J. , Troll Chris , Schwartz Camille , Naughton Colin , Ali Abdullah Mahmood , Raza Azra , Rao Varsha , Harkins-Kincaid Kelly , Green Richard Edward
Published: July 2021

DNA sequencing library preparation is a crucial step for next-generation sequencing of cell free DNA of plasma for cancer diagnostics. We present SRSLY, a robust single-stranded DNA library preparation method, that generates libraries with unique molecule identifiers (UMIs), with advantages over traditional double-stranded DNA preparations. Using 5 ng of plasma-derived cfDNA from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and gastrointestinal cancer cases, we prepared both SRSLY and double-stranded DNA (dsDNA) sequencing libraries with UMIs. The libraries were enriched using a Twist Biosciences custom hybridization probe set for an 800 kilobase cancer mutation panel. We sequenced to a panel depth of 1000x to 2000x, after correcting UMI sequences and removing duplicate reads. With comparable fold enrichment and on-target percentages, we observe complexities of 11-21 million unique molecules with SRSLY. This is approximately four times higher than dsDNA complexity of 2.8-4.9 million unique molecules. Using duplicate reads from the same unique molecule, we perform error correction on template read bases and are able to call low-variant allele frequency mutations. In particular, we show an increase in KRAS p.G12D variant allele fraction during progression from MDS to AML. SRSLY shows increased recovery of small fragments over dsDNA preparations and retains the native lengths and termini of all fragments. Apart from improved library complexity, we show that MDS and AML samples can be distinguished from healthy samples by their disrupted genomic profile of short to long fragments, which has been previously demonstrated in solid tumors but not for MDS or AML.

Citation Format: Charles J. Vaske, Chris Troll, Camille Schwartz, Colin Naughton, Abdullah Mahmood Ali, Azra Raza, Varsha Rao, Kelly Harkins-Kincaid, Richard Edward Green. Highly complex DNA sequencing library preparation for cfDNA enrichment panels using a single-stranded approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 556.
Abstract 558: Muti-gene ARMS PCR using both cfDNA and cfRNAin the supernatant of pleural effusion achieves rapid and accuracy driver gene mutations detection
Authors: Chen Xuejing , Li Kun , Liu Zichen , Gai Fei , Zhu Guanshan , Lu Shun , Che Nanying
Published: July 2021

Background Pleural effusion from patients with advanced non-small cell lung cancer has been proved valuable for molecular analysis, especially when the tissue sample not available. However, simultaneous detection of multiple driver gene alterations especially the fusions is still challenging.

Methods In this study, 77 patients with advanced NSCLC and pleural effusion were enrolled, 49 of whom had matched tumor tissues. Supernatants, cell sediments, and FFPE cell blocks were prepared from pleural effusion samples for detection of driver alterations by a PCR-based 9-gene mutation detection kit.

Results In addition to mutations in EGFR, KRAS and HER2 detected in genomic DNA from cell sediments, FFPE cell blocks, and in cfDNA from supernatants, and fusions in ALK detected in RNA from cell sediments and FFPE cell blocks, fusions in ALK were also successfully detected in cfRNA from supernatants. Compared with matched tumor tissue, the supernatant showed the highest overall sensitivity (81.3%), with 81.5% for SNV/Indels by cfDNA and 80% for fusions by cfRNA, followed by FFPE cell blocks (71.0%) and the cell sediments (66.7%). Within the group of treatment-naïve patients or malignant cells observed in the cell sediments, supernatant showed higher overall sensitivity (89.5%, 92.3%) with both 100% for fusions. Based on the results, an optimized driver gene mutations detection procedure of pleural effusion is proposed. With the proposed procedure, an overall sensitivity 85.3% was achieved, with 82.1% for SNV/Indels and 100% for fusions.

Conclusions CfDNA and cfRNA derived from pleural effusion supernatant (PES) have been successfully tested with a PCR-based multi-gene detection kit. An optimized procedure could maximize clinical value of testing pleural effusion samples and has good potential for routine clinical application.

Citation Format: Xuejing Chen, Kun Li, Zichen Liu, Fei Gai, Guanshan Zhu, Shun Lu, Nanying Che. Muti-gene ARMS PCR using both cfDNA and cfRNAin the supernatant of pleural effusion achieves rapid and accuracy driver gene mutations detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 558.
Abstract 564: BRAFV600 mutation in circulating tumor DNA can predict outcomes in early-stage melanoma
Authors: Gouda Mohamed Alaa , Polivka Jiri , Huang Helen , Treskova Inka , Pivovarcikova Kristyna , Meric-Bernstam Funda , Pesta Martin , Janku Filip
Published: July 2021

Background: Up to 70% of patients with resected high-risk melanoma develop disease recurrence within 5 years. Adjuvant immunotherapy or targeted therapy can reduce the recurrence rate below approximately 60%. It is, however, at the cost of possible toxicity including long-term side effects. We hypothesize that detection of plasma-derived circulating tumor DNA (ctDNA) from patients with resected melanoma can identify patients at high-risk of disease recurrence.

Methods: We developed an ultrasensitive and specific droplet digital PCR (ddPCR) - based method (Bio-Rad) to detect BRAFV600E-mutated ctDNA in pre-amplified cell-free DNA with sensitivity up to 2 mutant copies in the wild-type background. Plasma samples from patients with surgically resectable melanoma were collected before and after surgery and during follow-up visits for BRAFV600E ctDNA detection. Results obtained from ddPCR analysis were correlated with clinical outcomes.

Results: Total of 53 patients with resectable melanoma (29 males [54.7%]; 15 stage III [28.3%], 22 stage II [41.5%], 10 stage I [18.9%], 3 CIS [5.7%]; 25 with mutant tissue BRAF [47.2%]) and a median age of 60 years were included in the study. BRAFV600E-mutated ctDNA was detected in 25 patients (48%) before surgery and in 17 patients (32%) after surgery. Conversion of ctDNA BRAF status (from mutant to wild-type) in preoperative and postoperative samples occurred in 11 patients (21%). Patients with detectable ctDNA in samples collected after surgery had more disease recurrences (47.1% [n=8] vs. 20.6% [n=7]; P<0.05) and shorter disease-free survival (26 months vs unreached median survival; P<0.05) - and in the cohort of patients with confirmed BRAFV600E mutation in tumor tissue shorter overall survival (35 months versus unreached median survival; P<0.05). At all other studied follow up timepoints, there was no significant difference in outcomes between patients with BRAFV600E-mutated ctDNA and those without.

Conclusions: Our results demonstrate that ultrasensitive droplet digital PCR method can detect ctDNA in patients with resectable melanoma and that patients with detectable ctDNA in blood samples collected after surgery have inferior clinical outcomes.

Citation Format: Mohamed Alaa Gouda, Jiri Polivka, Helen Huang, Inka Treskova, Kristyna Pivovarcikova, Funda Meric-Bernstam, Martin Pesta, Filip Janku. BRAFV600 mutation in circulating tumor DNA can predict outcomes in early-stage melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 564.
Abstract 583: Analysis of solid pediatric tumor methylomes reveals a shared cancer recurrence signature in cell-free DNA
Authors: Buckley David N. , Tew Ben , Gooden Gerald , Triche Timothy , Salhia Bodour
Published: July 2021

Cancer is the second most common cause of death in children aged 1-14 years in the United States, with approximately 11,000 new cases and 1200 deaths annually. Due in part to the rarity and diversity of childhood tumors, a non-invasive diagnostic to identify patients at high risk of recurrence does not exist for patients with pediatric solid cancer. Cell-free (cf)DNA is rapidly becoming the standard for non-invasive screening, diagnosis, treatment and monitoring of human tumors. In particular, cfDNA methylation detection has proven to be a robust platform for “liquid biopsies,” with several important advantages over simple somatic variant detection. Here we present a cfDNA methylation signature derived from a pan-methylome analysis of recurrent pediatric tumors. The methylomes spanned 34 tumor tissue samples, 13 patient-matched adjacent normal, and 16 patient-matched plasma samples from 27 individuals, representing 11 different pediatric tumor types, including 10 neuroblastomas, 4 osteosarcomas and 2 hepatoblastomas. In addition, 13 adjacent normal samples and 15 normal plasma samples were used as DNA methylation controls. These samples were obtained from the Pediatric Oncology Experimental Investigators' Consortium (POETIC) under informed consent. DNA methylation was analyzed for all samples by whole genome bisulfite sequencing (WGBS). Using consensus DNA methylation calling, we identified a set of 402 differentially methylated regions (DMR) in genomic DNA from tissue, with substantial enrichment of hypomethylated DMRs across a majority of samples. CpGs that were commonly hypomethylated were combined into consensus ‘hotspots.' In order to discriminate between tumor/normal samples in tissue, we applied supervised machine learning to train a classifier on these 402 features. Mean methylation across these 402 CpG hotspots was significantly (p < 0.01) lower in disease plasma compared to normal control plasma samples. We then tested the classifier on the 16 patient-matched cfDNA samples and achieved a classification accuracy of 95% and an area under the curve (AUC) of 0.935. In summary, we have identified a promising set of DNA methylation features also detected in cfDNA that can potentially be used for recurrence risk stratification and measurable minimal residual disease monitoring in solid pediatric tumors.

Citation Format: David N. Buckley, Ben Tew, Gerald Gooden, Timothy Triche, Bodour Salhia. Analysis of solid pediatric tumor methylomes reveals a shared cancer recurrence signature in cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 583.
Abstract 620: Interrogating the molecular profile of lung cancer tumors in Puerto Rican Hispanics
Authors: Centeno-Girona Hilmaris , Montes-Rodríguez Ingrid M. , Rivera-Lynch Camila , Soto-Salgado Marievelisse , Rivera Noridza , Cruz-Correa Marcia
Published: July 2021

Background: In the United States, lung cancer accounted for approximately one-quarter of all cancer deaths; meanwhile, it was reported as the second cause of death in Puerto Rico (PR) for 2018. Worldwide, the development of new technologies such as next-generation sequencing (NGS) has allowed describing the profile of tumors and identifying mutation drivers in oncologic patients. The information on these is limited for Puerto Rican Hispanics (PRH).

Methods: Using retrospective data from CARIS Life Sciences from 2015 to 2020, we were able to estimate the prevalence of somatic mutations of 119 PRH with lung cancer. In addition, we compared them with the mutational profiles reported for lung cancer from the TCGA Pan-Lung data available in the cBioPortal for Cancer Genomics. Frequencies and percentages were calculated to describe the data.

Results: The results showed that the 10 most mutated genes for lung cancer tumors in PRH were TP53 (53.7%, n=67), EGFR (20.6%, n=73), KRAS (20.6%, n=73), T.M.L. (14.1%, n=64), APC (11.0, n=73), PTEN (10.3%, n=68), ARID1A (13.6%, n=44), PIK3CA (6.9%, n=73), STK11 (6.9%, n=72), and BRAF (5.5%, n=73). Additionally, only one positive mutation was found in MET (1.4%, n=72), and no mutations were identified in ALK and RET. When comparing to the top 10 tumor mutational profiles from the TCGA study, only BRAF was observed with a mutational frequency of 67.7% (n=1,444).

Conclusion: Understanding the common molecular pathways that affect the PRH population is necessary to develop new therapeutic modalities. Moreover, this preliminary review of oncogenic drivers in PRH with lung cancer will inform clinicians, scientists, and the health community about actionable mutations to guide research, treatment, and health policy efforts.

Citation Format: Hilmaris Centeno-Girona, Ingrid M. Montes-Rodríguez, Camila Rivera-Lynch, Marievelisse Soto-Salgado, Noridza Rivera, Marcia Cruz-Correa. Interrogating the molecular profile of lung cancer tumors in Puerto Rican Hispanics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 620.
Abstract 622: Gene panel-based immune profiling of human cancers
Authors: De Velasco Marco A. , Kura Yurie , Sakai Kazuko , Fujita Kazutoshi , Nozawa Masahiro , Yoshimura Kazuhiro , Nishio Kazuto , Uemura Hirotsugu
Published: July 2021

It is now well accepted that patients with inflamed tumors are more likely to benefit from immunotherapies targeting immune checkpoints such as CTLA4, PD1 and PD-L1.Several factors determine a tumor's immune status and identifying patients who may benefit from immunotherapy and determining which immune therapy to administer depends largely on the patient's tumor immunophenotype. We have developed an approach for immunoprofiling using a focused gene set (115 genes) and scoring system that assesses cell specific types and immune functions. This approach was developed in a preclinical cancer models and is being evaluated and tested in human cancers. We previously extracted data for the gene set and employed unsupervised network clustering to differentiate and classify cancer samples of patients from The Cancer Genome Atlas (TCGA) into immunologically distinct clusters. Here continue or examination and overview clinical features associated with individual clusters. A total of 8477 patients from the TCGA research network were profiled which included 28 solid cancer types. A total of 10 immune clusters (IC 1-10) were identified with a median cluster size of 971 patients with sizes ranging from 178 to 1570. All cancer types were represented in the ICs with mostly normal distribution except for IC 6 which was composed of 40% breast cancer patients and IC10 of which 1/5 of the cases were head-neck squamous cell carcinomas. ICs 2, 5 and 8 were associated with the poorest survival with median times at 73, 51 and 65 months, respectively. Conversely, patients in IC 9 experienced the longest overall survival, median time 148 months. The aggregate inflammation score, which measures overall tumor inflammation was positively correlated to overall survival (P=0.0109). IC 9 was correlated with the highest survival times and was positively associated with primary signatures related antitumor immunity such as high IFN gamma, cytotoxicity, and CD8 T cell and NK cell signatures. IC 8 was characterized with poor survival times and was associated with signatures related with immunosuppressive cells including granulocytic myeloid derived suppressor cells, M2 polarized macrophages and TH17 T cells, and an adenosine high signature. In addition, cases from this cluster also had low CTL signatures. Our findings show that gene expression data from a focused gene and our scoring algorithm could be used to differentiate and classify patients into various groups of immunologically distinct tumors types.

Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Gene panel-based immune profiling of human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 622.
Abstract 628: Ophtalmological manifestations of acute leukemias at university hospital center Yalgado Ouedrago
Authors: Millogo Kalo Roland , Kabore Delphine , Traore Solo
Published: July 2021

Acute leukaemias constitute 9.04% of malignant hemopathies (Burkina Faso, 2015). Their manifestations are mainly haematological, however other less frequent attacks are possible. We report here for the first time in Burkina Faso, three cases of ocular localizations in young adults. Observation 1: S.I, male, 17-year-old patient, student living in Ouagadougou with a history of recurrent sore throat and ear infections. He was admitted for asthenia and pallor in the context of polytransfusion. The myelogram showed a rich marrow marked by the presence of 15% blast cells. Ophthalmologic examination reported visual acuity of 1/10, stage II proptosis and lower chemosis, and massive retinal and macular hemorrhage. Chemotherapy could not be performed. he died a week later. Observation 2: TB, male, 23-year-old patient, welder, suffering from acute lymphoblastic leukemia diagnosed in front of a severe recurrent anemia and confirmed by a myelogram, showing a rich marrow infiltrated by lymphoblasts representing 47% of the medullary cells. The ophthalmologic examination revealed conjunctival-palpebral infiltration by acute leukemia with myogenic ptosis. The evolution was marked by an improvement in symptoms after a cure according to the Vincristine-Prednisone protocol and he was discharged from hospital weeks later. He was subsequently lost to follow-up. Observation 3: KK male, 25-year-old patient, artisanal miner residing in Ouagadougou with a history of peptic ulcer disease, carrier of acute myeloid leukemia (AML) confirmed by a myelogram. The ophthalmologic examination concludes with a bilateral inflammatory orthopathy with an exposure keratitis on site of AML. He received transfusion of concentrates of red blood cells and platelets, as well as corticosteroid therapy for 04 days. The following was marked by a rapid worsening of the symptoms and the death of the patient a week later. Conclusion: The extra-haematological manifestations of acute leukemia are rare. They are associated with a pejorative prognosis. This series of 03 cases that we report is the first described to date in our context.

Citation Format: Kalo Roland Millogo, Delphine Kabore, Solo Traore. Ophtalmological manifestations of acute leukemias at university hospital center Yalgado Ouedrago [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 628.
Abstract 639: Analysis of DNA minimal residual disease markers in pediatric solid cancers using quantitative real time PCR and droplet digital PCR
Authors: Subhash Vinod Vijay , Kamili Alvin , Erasmus Marie-Wong , Chen Dan , Huang Libby , Tyrell Vanessa , Atkinson Caroline , Venn Nicola , Cowley Mark , Marshall Glenn , Ekert Paul , Henderson Michelle , Sutton Rosemary , Norris Murray , Haber Michelle , Fletcher Jamie , Trahair Toby
Published: July 2021

Background: Minimal Residual Disease (MRD) detection is vital for therapy monitoring and relapse prediction in cancers. While RNA-based MRD assays are shown to be effective at diagnostic time-points, serial response monitoring does not provide additional predictive value.DNA-based assays may be more sensitive and allow longitudinal monitoring of the disease trajectory. This study aimed to develop MRD assays based on whole genome sequencing (WGS) data for high-risk neuroblastoma (HRNB) and Ewing sarcoma (ES) patients, to establish their sensitivity in quantitative PCR (qPCR) anddroplet digital PCR (ddPCR) formats, and to directly compare against RNA-based MRD assays.

Methodology: We analyzed WGS data to identify patient-specific chromosomal breakpoints in HRNB (N=6) and ES (N=6) patients, then established qPCR and ddPCR assays. Assay performance was validated using patient-derived cells spiked into bone marrow samples and in serially collected clinical samples as available. The DNA-MRD markers were compared against a panel of RNA-MRD markers for HRNB (TH/DCX/PHOX2B) and ES (EWS-FLI1/ERG/ETV1).

Results: DNA-MRD markers showed high sensitivity and quantitation ranging from 10-4(0.01%) to 10-5(0.001%) in qPCR assays and ddPCR assays. In clinical samples of HRNB and ES, DNA-MRD markers were detected in diagnosis specimens and predicted disease trajectory. Among RNA-MRD markers, TH & EWS-FLI1 transcripts showed the maximum detection sensitivity (0.01%), however was less sensitive than the patient specific DNA-MRD markers.

Conclusion: This study identifies DNA-MRD analysis as a reliable complimentary approach to RNA-MRD assays and highlights its enhanced sensitivity and clinical utility for monitoring treatment response and disease progression in solid pediatric tumors.

Citation Format: Vinod Vijay Subhash, Alvin Kamili, Marie-Wong Erasmus, Dan Chen, Libby Huang, Vanessa Tyrell, Caroline Atkinson, Nicola Venn, Mark Cowley, Glenn Marshall, Paul Ekert, Michelle Henderson, Rosemary Sutton, Murray Norris, Michelle Haber, Jamie Fletcher, Toby Trahair. Analysis of DNA minimal residual disease markers in pediatric solid cancers using quantitative real time PCR and droplet digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 639.