bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative assessment of different radiological criteria to identify paradoxical hyperprogression (HPD) to IO drugs.
Authors: Gomes de Morais Ana Luiza , Cardenas Jose M , de Miguel Luken Maria Jose , Boni Valentina , Moreno Irene , Ao Gerileto , Liu Run Han , de Hoyos Fernando Bergaz , Cubillo Antonio , Calvo Emiliano
Published: May 2020

e15229

Background: HPD is a new pattern of response consisting of accelerated tumor growth due to immunotherapy (5%-20% of pts on IO drugs). The tumor growth rate (TGR, Champiat et al.) estimates the differential increase in tumor volume over time, pre- and on IO drugs, to assess for HPD, but it is not easily calculated in practice. A comparison of the different methods to identify HPD is missing. Methods: Retrospective study of 182 consecutive pts treated in Phase 1 studies of IO drugs at START Madrid-CIOCC in 2017-8, comparing 3 HPD measurement criteria (Champiat, Matos -which does not use pre-baseline CT scans-, and Saâda-Bouzid). Cohen’s Kappa index was calculated as a measure of the agreement between the 3 methods, being those values closer to 1 the more concordant ones. Overall survival (OS) rates were calculated by Kaplan-Meier (p < 0.05 to be significant). Results: 99 (54%) of 182 pts had progressive disease at cycle 1 of treatment and in 62 (34%) pre-baseline CT scans were available to calculate TGR. The Champiat method identified 18 (9.9%) pts with HPD. Of 61 cases validated to comparison, the Matos criteria labeled 27 pts (14.8% of 182) with HPD, of whom only 10 pts coincided with those identified by Champiat with a low agreement (Kappa: 0.140, p:0.251). No significant differences in OS between pts with non-HPD vs HPD by Matos criteria were seen (p = 0.16). With Saâda-Bouzid method, of 59 cases validated to comparison, 17 pts (9.3% of 182) were labeled with HPD and 13 of them coincided with those identified by Champiat method with a high agreement (Kappa:0.706, p:0.000). Differences in OS between non-HPD vs HPD pts were statistically significant (p = 0.038). Conclusions: HPD might have a detrimental effect to 10% of pts on IO drugs, also in our series. Every effort should be done to obtain pre-baseline CT scans to identify HPD response to IO drugs, based on differential TGR. The use of Saâda-Bouzid method is preferred due to its practical application and its correlation with survival. [Table: see text]
High correlation between TMB, expressed TMB, and neoantigen load using tumor: Normal whole exome DNA and matched whole transcriptome RNA sequencing.
Authors: Szeto Christopher , Gounder Mrinal M. , Parulkar Rahul , Nguyen Andrew , Rabizadeh Shahrooz , Reddy Sandeep K.
Published: May 2020

e15238

Background: Tumor Mutation Burden (TMB) has emerged as a biomarker of response to immune checkpoint inhibitor therapy, independent of PDL1 expression. The optimal cutoff for high versus low TMB is unknown and neoantigen load has been proposed as an alternative biomarker. We have previously described neoantigen silencing at the RNA level as potential means of immune escape. We hypothesized that expressed TMB (eTMB) and neoantigen load would differ from TMB. Methods: Using data from matched tumor:normal DNA, whole exome sequencing at 150x average depth, and whole RNA transcriptome at ~200x106 reads per tumor from a commercial database of 1395 heterogeneous cancer patient samples, we measured TMB and eTMB ( > 2 alternate RNA reads supporting). Neoantigen load was measured as the count of unique 9mer peptides resulting from expressed somatic-specific variants with predicted binding affinity < 500nM by NetMHC. Results: High correlation between TMB and eTMB was seen (R2 = 0.94). No significant differences were seen between TMB, eTMB, and predicted neoantigen load. Using a cutoff of 200 non-synonymous variants (Rizvi 2015) to define TMB high versus highest quintile (TMB-20) (Samstein 2019) we saw high agreement of the 2 methods to assign TMB status as high v low. In the TMB-20 group with low TMB ( < 200), most often reclassified were soft tissue sarcoma (18%) and pancreatic cancers (17%). Conclusions: Surprisingly, eTMB, TMB-20, and neoantigen load showed no significant difference from TMB using 200 variants as a cutoff for high. Tissue-specific TMB may be useful in patient with Sarcoma and Pancreatic cancers. Additional data inputs such as microbiome, chemokine expression, and TME cell phenotyping may be required to improve upon TMB as a biomarker of immunotherapy response.
Cross-site concordance evaluation for the CIMAC-CIDC network to establish QC metrics enabling reliable generation of WES and RNA-seq data.
Authors: Wistuba Ignacio Ivan ,   CIMAC-CIDC Network
Published: May 2020

e15240

Background: Whole-exome sequencing (WES) and RNA-sequencing (RNA-seq) have become key components of cancer genomics, and analysis to provide comprehensive evaluation of spectrum somatic alterations and changes in gene expression present in cancer samples. To test the ability to generate comparable data for these genomic assays across CIMAC-affiliated sequencing centers, the CIDC performed a cross-site harmonization effort at two CIMAC sites (MD Anderson [MDACC] and DFCI/Broad Institute) and the MoCha Lab (MoCha) at the National Cancer Institute. Fresh frozen (FF) and FFPE samples from 8 non-small cell lung carcinoma (NSCLC) tumors were provided by MDACC, and processed for DNA and RNA extraction, aliquoted, and sent by MDACC to MoCha, MDACC and DFCI/Broad to be processed as replicates. Two reference samples,pools of 10 HapMap cell lines were sent for WES processing. Methods: WES analysis was carried out using center-specific pipelines as and central workflows that included: coverage metrics (target-based and genome-wide), variant calls (somatic and germline), variant annotations, tumor purity and clonality. Only minimal differences in somatic mutation calls were found between different replicates and centers for the HapMap samples, and technical differences were much smaller than biological differences between the patient tumors. For both sets of data, when focusing on reads at > 50X common coverage and events with variant allele frequency (VAF) > 5%, the different centers achieved high precision ( > 98%) and recall ( > 87%).Further, ISA for patient samples ranged from 0.667-0.875, while that for HapMap samples ranged from 0.817-0.827. Results: The RNA-seq analysis pipeline used the tools STAR for read mapping, RSEQC for quality control, SALMON for transcript quantification and LIMMA for batch removal on log transformed TPM values. Our analysis revealed that a DV200 of 24- 30% was a reasonable cutoff to achieve consistent quality in RNA-seq data generation. In addition, a medTIN cutoff value of 50% was found to be effective in determining the quality of the samples. Using these criteria, the Spearman correlation of log(TPM) among all the FF samples from the same tumor were higher than 0.976, and among all FFPE samples from the same tumor were higher than 0.942. Conclusions: In summary,cross-center concordance evaluation has identified the factors that introduce variability in the analyses, while also demonstrating the ability to provide comparable results across CIMAC sites by using a centralized analysis pipeline.
Analytical validation of iSort digital cytometry for leukocyte enumeration in clinical tumor specimens.
Authors: Newman Aaron M. , Nakao Aki , Li Kathy , Wilson Douglas Collins , Liu Chih-Long , Diehn Maximilian , Alizadeh Ash A.
Published: May 2020

e15243

Background: Tumor infiltrating leukocytes (TILs) are major determinants of anti-tumor immune responses and mediate effector functions for diverse immunotherapies. Techniques relying on intact cells such as flow cytometry (FACS), immunohistochemistry (IHC), and scRNA-Seq can be powerful for measuring TIL heterogeneity. However, such methods are often impractical on large patient cohorts, can be poorly quantitative, and some cannot be applied to formalin-fixed paraffin embedded (FFPE) specimens. We developed iSort, a transcriptome deconvolution method based on CIBERSORTx for TIL enumeration. We evaluated iSort by comparing its performance in FFPE and fresh frozen (FF) human tumor tissues to established methods. Methods: We profiled human tumors for diverse TIL subsets, as quantified by iSort, FACS, and IHC. iSort was performed on RNA-Seq libraries derived from bulk human tumors, including FF tumor tissue samples, tumor cell suspensions obtained after dissociation, and FFPE specimens. We assessed the influence of tissue fixation on common TIL subsets enumerated by iSort, by comparing matched pairs of FF and FFPE lung tumor specimens (NSCLC). Finally, we compared iSort with automated microscopic enumeration of CD8 TILs by IHC on primary human lung tumors (FFPE/NSCLC). Results: iSort TIL enumeration was highly correlated with FACS across primary human tumors (CRC = 3, HNSCC = 1) for common TIL subsets (B cells, CD8 T, CD4 T, monocytes/macrophages, dendritic cells, PMNs/MDSCs) and total immune content (median r = 0.91 [0.71 - 0.99]). Tissue dissociation caused significant depletion of monocytes/macrophages (p < 0.01), when comparing bulk tumor specimens with cell suspensions after sample digestion. iSort showed high correlation across 12 TIL subsets when comparing 9 pairs of FF and FFPE specimens (r = 0.90). Finally, we observed high linearity between iSort and microscopic enumeration of CD8 TILs from IHC (r = 0.87, n = 5). Conclusions: iSort digital cytometry achieves accurate and robust TIL enumeration as compared with FACS and IHC, showing comparable performance in fixed and frozen primary human tumors. Importantly, iSort is directly applicable to bulk tumors and is unaffected by tissue dissociation artifacts, unlike methods requiring cell suspensions for TIL enumeration (FACS, scRNA-seq). Therefore, iSort is a promising approach for development and measurement of immunotherapy biomarkers.
A survey of cancer testis antigen (CTA) expression across T-cell lymphoma subtypes.
Authors: Ma Helen , Soderquist Craig R. , Marchi Enrica , Scotto Luigi , Bhagat Govind , O'Connor Owen A.
Published: May 2020

e15246

Background: T-cell lymphomas (TCL) are a rare and heterogeneous group of non-Hodgkin lymphomas (NHL) that originate from T- and NK-cells and portends a poor prognosis. One promising therapeutic strategy involves generation of tumor associated antigen directed T-cells (TAA-T) against specific CTA. RNAseq have established that combinations of histone deacetylase inhibitors and hypomethylating agents uniquely induce genes characterized as cancer testes antigens (CTA), which may increase immunogenicity. There is a lack of data on the baseline expression of CTA across the TCL, limiting application of TAA-T. We sought to establish the baseline expression of CTA in PTCL using immunohistochemistry to better understand treatment effects. Methods: A tissue microarray was created using formalin-fixed paraffin embedded tissue from 40 patients with diverse T-cell neoplasms. Expression of WT1, SCP1, survivin, PLAC1, SSX2, and PRAME was evaluated by immunohistochemistry. The percentage of positive tumor cells (divided into quartiles), staining intensity (weak, moderate, strong), and subcellular localization (nuclear, cytoplasmic) was assessed. Results: Forty patients with T-cell lymphomas were included in the analysis. The median age of diagnosis and median number of treatments was 60.2 years and 2 (0-9), respectively. Thirty-nine (98%) patients expressed at least one CTA. A high frequency of survivin, which is associated with cell proliferation and is expressed on normal lymphoid tissue, was expressed in 98% of TCL, 5% expressed WT1, and 12.5% expressed SCP1, and expression of other CTA was not observed. Of five patients with anaplastic large cell lymphoma, 3 samples were positive for SCP1 and one was also positive for WT1. One of 9 angioimmunoblastic T-cell lymphoma samples was positive for SCP1. Of 4 T-lymphoblastic lymphoma patients, 1 was positive for WT1, and of 3 transformed mycosis fungoides specimens, 1 demonstrated SCP1 expression. In this sample set, we did not find a correlation with expression and response to chemotherapy. There were no patients with CTA expression who were treated with epigenetic treatments. Conclusions: We described low expression of CTA on T-cell lymphomas, which is consistent with the literature on expression by RT-PCR. By establishing this baseline expression of CTA, we have the foundation on which to measure changes in CTA expression pre- and post-treatment by immunohistochemistry.
Developing and validating an approach for diagnosing and prognosticating cancer from biochemical motifs in T-cell receptors.
Authors: Ostmeyer Jared L , Cowell Lindsay G , Christley Scott
Published: May 2020

e15260

Background: Immune repertoire deep sequencing allows profiling T-cell populations and enables novel approaches to diagnose and prognosticate cancer by identifying T-cell receptor sequence patterns associated with clinical phenotypes and outcomes. Methods: Our goal is to develop a method to diagnose and prognosticate cancer using sequenced T-cell receptors. To determine how to profile the specificity of a T-cell receptor, we analyze 3D X-ray crystallographic structures of T-cell receptors bound to antigen. We observe a contiguous strip typically 4 amino acid residues in length from the complimentary determining region 3 (CDR3) lying in direct contact with the antigen. Based on this observation, we extract 4 residue long snippets from every receptor’s CDR3 and represent each snippet using biochemical features encoded by its amino acid sequence. The biochemical features are combined with information about the abundance of the snippet or the receptor and scored using a machine learning based approach. Each predictive model is fitted and validated under the requirement that at least one positively labelled snippet appears per tumor and no positively labelled snippets appear in healthy tissue. Results: Using a patient-holdout cross-validation, we fit predictive models to distinguish: 1. colorectal tumors from healthy tissue matched controls with 93% accuracy, 2. breast tumors from healthy tissue matched controls with 94% accuracy, 3. ovarian tumors from non-cancer patient ovarian tissue with 95% accuracy (80% accuracy on a blinded follow-up cohort) 4. and regression of preneoplastic cervical lesions over 1 year in advance with 96% accuracy. Conclusions: Immune repertoires can be used to diagnose and prognosticate cancer.
A comparative study of PD-L1 IHC assays using immune cell scoring and CPS in breast cancer.
Authors: Li Bin , Dillon Lloye M. , Jones Jennifer , Desai Keyur , Gold David , Pandya Dimple , Roberts Mustimbo
Published: May 2020

e15262

Background: The clinical benefit of immune checkpoint inhibitors (ICIs) targeting the programmed death-1/programmed death ligand 1 (PD-L1) pathway has previously been demonstrated across a range of tumor types, including in PD-L1+ patients with metastatic triple-negative breast cancer (TNBC). Various PD-L1 immunohistochemistry (IHC) assays and scoring algorithms are being investigated to select patients with breast cancer (BC) most likely to respond to ICIs. Scoring algorithms include PD-L1 expression on tumor cells, immune cells (ICs), or both. We compared the analytical concordance of 3 PD-L1 IHC assays and evaluated PD-L1+ prevalence, using combined positive score (CPS) and % IC scoring algorithms in commercially procured TNBC and hormone receptor–positive, HER2-negative (HR+/HER2−) BC samples. Methods: PD-L1 expression was assessed by HistoGeneX (Naperville, IL) in 163 commercially procured, surgically resected, formalin-fixed, paraffin-embedded BC samples (mostly stage I–III) using the Ventana PD-L1 (SP142) and Dako PD-L1 IHC 28-8 and 22C3 pharmDx assays in conjunction with the CPS (28-8 and 22C3 assays) and % IC algorithms (SP142 and 28-8 assays). PD-L1+ prevalence with each assay and concordance between assays were calculated using CPS ≥ 1 and IC ≥ 1% cutoffs, with a single pathologist assigned to each scoring algorithm. Results: 93 HR+/HER2− BC and 70 TNBC samples were evaluable for PD-L1 expression across all assays and algorithms. Overall concordance was higher between the 28-8 and 22C3 assays (CPS cutoff of 1) than between the 28-8 and SP142 assays (IC cutoff of 1%). PD-L1+ prevalence was similar with the 28-8 and 22C3 assays (CPS ≥ 1) and higher with the 28-8 assay (IC ≥ 1%) than with the SP142 assay (IC ≥ 1%). PD-L1+ samples identified by the SP142 assay (IC ≥ 1%) were mostly included within PD-L1+ sample sets defined by the 28-8 assay (IC ≥ 1% and CPS ≥ 1). PD-L1+ prevalence was higher in TNBC vs HR+/HER2− BC for all assays (Table). No clear trend in PD-L1+ prevalence was observed across tumor grade and stage. Conclusions: High analytical concordance was observed between the 28-8 and 22C3 assays (CPS cutoff of 1) in both HR+/HER2− BC and TNBC samples. PD-L1+ prevalence varied according to IHC assay, scoring algorithm, and cutoff used. Further studies are needed to select the most appropriate PD-L1 assay and scoring algorithm for BC clinical trials. [Table: see text]
Phase I trial of personalized mRNA vaccine encoding neoantigen in patients with advanced digestive system neoplasms.
Authors: Zhan Xianbao , Wang Bin , Wang Yiran , Chen Longpei , Peng Xiaobo , Li Jie , Wu Meihong , Zhang Linli , Tang Shuhui
Published: May 2020

e15269

Background: Cancer cells have characteristics of genetic instabilities and accumulate somatic mutations rapidly which could produce tumor specific antigens (TSAs) called as neoantigens. As the cancer vaccine based on TSAs has potential to treat the disease, personalized neoantigen-based immunotherapies are emerging. We report for the first time that vaccines are used to treat advanced digestive system neoplasms. Methods: In this single-arm, open-label, first-in-human phase I pilot study (NCT03468244) to investigate the feasibility, safety, immunogenicity and clinical activity of personalized mRNA vaccine. Patients with advanced digestive system neoplasms received up to 20 stimulatory synthetic long peptides vaccine at a dose of 0.2 mg on days 1 and 0.8mg on days 2, administered subcutaneously of every 3 weeks(Q3W) for 12 weeks. Patients was treated with standard treatments according to NCCN guideline simultaneously. Adverse Event (AE) grade categorization is according to CTCAE 4.0, and tumor response was assessed per RECIST 1.1. Immune factors and ELISPOT assay were examined every 3 weeks after vaccination. Results: As of February 1st, 2020, three patients (52yrs, 47yrs and 52yrs; ECOG PS: 0) with advanced rectal, colon and gastric cancer completed the vaccine therapy for 3-4 cycles. There were no serious adverse events, treatment-related death or severe neurotoxicity occurred in the study. The most common treatment-emergent adverse event (TEAE) was erythema at injection sites in three patients. Vaccine combined standard treatments treatment was associated with G3/4 hematological side effects: leucopenia (18%); neutropenia (9%); anemia (0); thrombocytopenia (18%). Blood samples for immune monitoring (ELISPOT and intracellular cytokine staining [ICS]) were collected pre-dose and at weeks 3 and 12. The level of IL-1β, IL-2 receptor, IL-6, IL-8, IL-10, and TNF-α increased for all patients. Especially, IL-8 and IL-2R increased significantly for more than 800 times in the patient with rectal cancer and more than 4 times in the patient with gastric cancer after injection, respectively. The PFS were 3.2, 3.0 and 3.0 months and the OS were 9.1, 6.0 and 7.8 months, respectively. Best response was 2 SD and 1 PD. Conclusions: This clinical study indicated that personalized mRNA vaccine may be safe and could activate immune response in vivo which is convinced by ELISPOT assay in vitro. Further studies are indicated to explore the personalized mRNA vaccine in gastrointestinal cancer. Clinical trial information: NCT03468244 .
Alterations of Raman profiles of nasopharyngeal carcinoma cells after treated with chemodrugs detected by laser tweezer Raman spectroscopy.
Authors: Li Miaomiao , Chen Xiaochuan , Lin Ting , Huang Zongwei , Wu Shihong , Liu Wei , Lin Duo , Zhang Lurong , Qiu Sufang
Published: May 2020

e15513

Background: To explore the metabolic alterations of nasopharyngeal carcinoma (NPC) cells after treated with chemodrugs, the Raman profiles were characterized with laser tweezer Raman spectroscopy. Methods: Two NPC cell lines (CNE2 and C666-1) were treated with gemcitabine, cisplatin, and paclitaxel, respectively. The high-quality Raman spectra of cells without or with treatments were recorded at the single-cell level with label-free laser tweezers Raman spectroscopy (LTRS) and analyzed for the differences of alterations of Raman profiles. Results: Tentative assignments of Raman peaks indicated that the cellular specific biomolecular changes associated with drug treatment, including changes in protein structure (e.g. 1655 cm−1), changes in DNA content and structure (e.g. 830 cm−1), destruction of DNA base pairs (e.g. 785 cm−1), and reduction in lipids (e.g. 970 cm−1). Besides, both principal components analysis (PCA) combined with linear discriminant analysis (LDA) and the classification and regression trees (CRT) algorithms were employed to further analyze and classify the spectral data between control group and treated group, with the best discriminant accuracy of 96.7% and 90.0% for CNE2 and C666-1 group treated with paclitaxel, respectively. Conclusions: This exploratory work demonstrated that LTRS technology combined with multivariate statistical analysis has promising potential to be a novel analytical strategy at the single-cell level for the evaluation of NPC-related chemotherapeutic drugs.
A retrospective analysis of venous thromboembolism trends in chemotherapy-induced anemia.
Authors: Bryer Emily Jane , Kallan Michael J. , Chiu Ting-Shan , Scheuba Katharina M. , Henry David H.
Published: May 2020

e15515

Background: Anemia is a common and unfortunate consequence of chemotherapy. Patients receiving a variety of chemotherapy regimens often develop chemotherapy–induced anemia (CIA), which contributes to poor outcomes, including increased mortality. Prompt and effective treatment of CIA is essential to prevent fewer chemotherapy dose delays and reductions. Despite the baseline coagulopathies present in patients with malignancy, administration of RBC transfusions is associated with venous thromboembolism (VTE). It remains unknown whether the risk of VTE in patients with CIA is greater among patients who receive single or multiple RBC transfusions. Methods: A retrospective study analyzed 10,269 University of Pennsylvania Health System patients with malignancies of various type, stage, and histopathology who developed CIA between 5/1/08-12/31/17. We analyzed rates of VTE development after adjusting for RBC intervention with a 95% confidence interval during the 90 days following CIA diagnosis. Results: Among the 10,269 patients with CIA, 2,008 patients (19.6%) received only RBC transfusion as anemia treatment; of that group, 545 (27.1%) developed a VTE. Transfusion of 2+ units RBC compared with 1 unit of RBC was associated with an increase in VTE development (RR = 1.37, 95% CI 1.09-1.72). The relationship of higher VTE development with increasing number of transfusions is displayed below. Conclusions: While RBC transfusion in patients with CIA is independently associated with VTE, our data further suggests an exponential risk of VTE development with increasing numbers of RBC transfusions. [Table: see text] [Table: see text]
Effect of different dox concentration with hapten and dose selection intratumoral injection on the survival time of tumor bearing mice.
Authors: Fu Qiang , Han Yan , Yu Baofa ,   Shandong Baofa Cancer Therapeutics, Inc,
Published: May 2020

e15517

Background: To explore the effect of chemotherapeutic drug (Dox) with the concentration on the survival of tumor-bearing mice after intratumoral injection plus hapten. It is important to observe whether there is any difference in survival rate between the different dose of drug-administered group with hapten and the positive control group. Methods: The H22 liver tumor-bearing mice with the longest path length of tumors of 5-8 mm were selected and administered according to the tumor volume. The second dose was administered at intervals of 7 days. The death of the mice was observed daily and the time of death was recorded in order to evaluate the survival of the mice. Results: The effect on body weight, except for the negative control group, decreased in weight after the first administration, and the effect was reversible. The effect on tumor volume was significantly slower than that of the negative control group after administration. In the life extension rate study, the sustained-release drug group was compared with the corresponding positive control group, the sustained-release drug group II and the positive control group II, the sustained-release drug group IV group and the positive control group IV group, and the sustained-release drug group V and The positive control group V had significant significance (P < 0.01). Therefore, the survival time of the group IV of the sustained-release drug was better than that of the positive control group IV, and the dose of the group IV of the sustained-release drug was the optimal dose. Conclusions: The reduction of doxorubicin hydrochloride concentration can prolong the survival time of mice and the survival rate of the sustained-release drug group with hapten is generally higher than that of the positive control group. By comparing the tumor inhibition rate and survival test results of the slow release drug group with hapten and the positive control group, the concentration of doxorubicin hydrochloride was initially screened at 1.0 mg/ml which is best concentration for this therapy.
Can tumor cells be distinguished from blood cells by mechanical parameters? A label-free CTC detection approach.
Authors: Nel Ivonne , Morawetz Erik W , Kaes Josef A , Aktas Bahriye
Published: May 2020

e15526

Background: Even years after successful treatment of the primary tumor about 30% of breast cancer patients are suffering from metastatic relapse. One reason might be hematogenous spread. Therefore circulating tumor cells (CTC) in the blood might be interesting and easy accessible surrogate markers for disease monitoring. Due to phenotypical, mechanical and functional changes during cancer progression and treatment response, isolation of CTC subpopulations remains very challenging. Here we focused on the characterization of CTC mechanics to evaluate the utility of mechanical parameters for CTC separation from blood. Methods: For the first time, we investigated the active and passive mechanical CTC properties such as elasticity, viscosity and contractile force exertion using a dual beam trap. For proof of premise we used peripheral blood mononuclear cells (PBMC) from a healthy donor and human GFP-expressing MDA-MB 231 breast cancer cells to mimic a CTC model system and to measure cell type specific mechanical deformation profiles using an optical rheometer. For translational experiments blood samples were collected from patients with mamma or vulvar carcinoma. Hematopoietic cells were depleted using CD45. The remaining cell suspensions were applied to the optical stretcher and rheological parameters were measured in the same manner. Results: We were able to reveal distinct mechanical profiles from hematopoietic cells and breast cancer cells, respectively. The optical deformability was significantly different in both the model and the translational, systems comparing healthy PPMC to MDA-MB 231 cells, respective possible CTC candidates from breast and vulvar carcinoma patients. Evaluation of secondary parameters like viscosity and activity using a machine learning algorithm allowed for a clear distinction between hematopoietic and cancer cells in the model system. CD45-positive and -negative cells from patients with mamma or vulvar carcinoma (n = 7) showed distinct differences in the rheological behavior of both cell populations. Surprisingly, the mechanical profiles of CD45-negative cells from mamma carcinoma samples were severely different from vulvar carcinoma. Conclusions: Together with morphology and cell size, the cellular deformation pattern might be a proper tool for marker-free CTC detection in the peripheral blood of breast cancer patients.
Clinical utility of circulating genetically abnormal cells within low-dose computed tomography lung cancer screening: A correlative MCPND trial study.
Authors: Feng Mingxiang , Lin Miao , Zhou Haining , Zhu Yunhe , Chen Baishen , Ye Xin , Huang Chuoji , Zhang Juncheng , Bai Chunxue
Published: May 2020

e15536

Background: Recent screening trial results using low-dose computed tomography (LDCT) have been shown to reduce lung cancer mortality in high-risk patients. However, high false-positive rates, inter-grader variability, and radiation injuries are challenging, and therefor highlight the necessity for complementary biomarkers. The diagnostic performance of circulating genetically abnormal cells (CACs) was respectively evaluated in samples collected from patients with pulmonary nodules before and after lung lobectomy within the Multicenter Chinese Pulmonary Nodule Detection (MCPND) trial. We also propose a deep learning that detects related genetically abnormal cells in excisions of symptomatic lesions to identify the source of CACs. Methods: 269 plasma samples (n = 179, before surgery; n = 90, after surgery) from 179 participants, including 125 patients with lung cancer and 54 donors with benign nodular lesions, were analyzed by using a FISH–based assay for 3p22.1, 3q29, 10q22_3 and CEP10. Diagnostic performance of CAC was evaluated in a blinded validation group. 109 paraffin-embedded specimens (45 benign nodular lesions, 64 malignancies) obtained from participants were tested in the same way to identify the genetically abnormal cells. Results: The diagnostic performance of CACs for lung cancer detection was 91% for sensitivity and 81% for specificity. For all participants, CACs had a positive predictive value of 92% and 80% for negative predictive value, respectively. Area under the ROC curve was 0.89 (P < 0.01; 95%CI (0.82, 0.96)). 90 plasma samples were drawn from patients within 10 days after surgery. Of these samples, 58 participants’ CACs level reduced and 25 of them remained stable. Moreover, of 64 malignant specimens, genetically abnormal cells were detected in 60 specimens. However, only 4 specimens were detected having genetically abnormal cells in 45 benign nodular lesions. Conclusions: This validation study indicated that the number of genetically abnormal cells in circulation decreased after surgical resection, which suggested CACs could be released from lung cancer. CACs have diagnostic and prognostic value and could assist to improve the diagnostic accuracy in early stage lung cancer after low-dose computed tomography. Clinical trial information: ChiCTR1900026233.
Potentially targetable alterations identified in circulating tumor DNA (ctDNA) from patients (pts) with advanced rare cancers.
Authors: Okuma Hitomi Sumiyoshi , Yonemori Kan , Kojima Yuki , Tanioka Maki , Sudo Kazuki , Noguchi Emi , Hijioka Susumu , Wakakuwa Keiko , Kato Ken , Ichikawa Hitoshi , Hirakawa Akihiro , Kuchiba Aya , Shibata Taro , Sukigara Tamie , Nakamura Kenichi , Yamamoto Noboru
Published: May 2020

e15540

Background: People with rare cancers have limited treatment options. In April 2017, the MASTER KEY Project, a prospective registry study with multiple clinical trials embedded, was established to promote treatment development for rare cancers. ctDNA analysis by next-generation sequencing (NGS) has provided new insight into personalized medicine; however, reports are focused on common cancers. Using our platform, we examined the feasibility of ctDNA NGS analysis for rare solid cancers. Methods: Prospectively consented pts with advanced rare cancers enrolled in MASTER KEY had ctDNA NGS testing (Guardant360, Guardant Health). Alterations were assessed for incidence according to cancer type, functional impact, and therapeutic implications. Results: From Nov 2018 to Jan 2019, 98 samples from 100 consented pts were analyzed. Diseases included soft tissue sarcoma (39); cancers of the ovary (12) and salivary gland (9); neuroendocrine tumors (8); carcinoma of unknown primary (6); carcinosarcoma (5); mesothelioma (4); gastrointenstinal stromal tumor (3); and olfactory neuroblastoma (2). All pts were Japanese, male/female = 40/58, median age 53, ECOG performance status 0/1/2/3 = 56/41/0/1, prior pharmacotherapy yes/no = 77/21. 76% of pts (75/98) had at least 1 alteration detected (median 2.8/pt; range: 0-9), with variant allelic frequency of 0.1-73.3%; 61%(60/98) of pts had a likely pathogenic variant, most commonly TP53 (43), KRAS (11), and NRAS (6); 36% (36/98) had a potentially actionable variant, such as BRCA1/2 (6), EGFR (6), PIK3CA (6), ARID1A (5), FGFR1/2/3 (4), and other homologous recombination deficiency genes. Known resistant mutations such as ROS1 G2032R and RAS mutations were detected in patients on a ROS inhibitor and a BRAF inhibitor, respectively, which suggested drug resistance prior to clinical diagnostic imaging. Mean turn-around-time from blood collection was 9.5 days. Conclusions: ctDNA testing is capable of rapidly identifying alterations that lead to clinical decision making in pts with advanced rare cancers, who have a crucial lack of treatment options compared to common cancer pts. Clinical trial information: UMIN000034394.
Monitoring of therapeutic efficacy to CDK4/6 inhibitors and early detection of metastatic relapse in breast cancer by ultra-deep sequencing of plasma cell-free DNA.
Authors: Chin Yoon Ming , Shibayama Tomoko , Otaki Masumi , Chan Hiu Ting , Ono Makiko , Ito Yoshinori , Takahashi Shunji , Ohno Shinji , Ueno Takayuki , Nakamura Yusuke , Low Siew-Kee Kee
Published: May 2020

e15544

Background: CDK4/6 inhibition substantially improves progression-free survival for women with advanced estrogen receptor-positive breast cancer. Despite this, most patients experience acquired resistance. The analyses of cancer specific genomic aberrations in plasma cell free DNA, or commonly referred to as ‘liquid biopsy’, has generated immense interest. The role of liquid biopsy enables monitoring of therapeutic efficacy to detect relapse in a minimally invasive manner. It would drastically impact risk stratification, treatment selection and patient clinical outcome in advanced stage breast cancer. Methods: We have recruited consecutive patients with metastatic breast cancer who received CDK4/6 inhibitor treatment in combination with endocrine therapy between April 2018 and December 2019. Somatic mutations identified by the Oncomine Pan-Cancer cell free assay were used to assess the mutation profiles of circulating tumor DNA (ctDNA) by ultra-deep sequencing (~56,000 depth) of the plasma DNA. Plasma samples (N = 146) were collected before and during treatment (2 years) or until disease progression. Paired white blood cells (WBC) were also sequenced to exclude clonal hematopoiesis-derived mutations. Results: Twenty-eight patients were enrolled. Based on RECIST criteria, 1 patient showed complete response (CR) within 18 months of treatment, 5 patients showed partial response (PR) 6-18 months from start of treatment, 9 patients showed stable disease (SD) condition within 3-18 months and 13 patients developed progressive disease (PD) within 1-12 months. In 22 of 28 patients (79%), ctDNA was detected prior to start of treatment (12 responders; 10 PD) and used as a monitoring marker. Majority of mutations detected in pre-treatment samples were TP53, PIK3CA and ESR1. Among the 12 ctDNA positive responders, ctDNA levels of 9 patients were undetectable within 2 weeks to 6 months from start of treatment (median 3 months). ctDNA profiles were more reflective of clinical response compared to the tumor markers CEA and CA15-3. In 7 of the 10 PD patients with ctDNA monitoring markers, ctDNA showed higher sensitivity to predict disease progression compared to tumor markers that remained stagnant or below normal threshold. ctDNA also predicted PD earlier than radiological images with a median lead time of 3 months. Conclusions: ctDNA assessment for therapy monitoring and early relapse detection in advanced breast cancer is feasible and provides a basis to evaluate early therapeutic interventions.
Urinary cell-free DNA analysis for tumor mutation detection in patients with oligometastatic colorectal cancer.
Authors: Pellini Bruna , Pejovic Nadja , Harris Peter , Feng Wenjia , Usmani Abul , Qaium Faridi , Fields Ryan , Chaudhuri Aadel
Published: May 2020

e15545

Background: Cell-free DNA (cfDNA) analysis from blood is a useful liquid biopsy strategy. However, it requires phlebotomy and moderate volumes of plasma. Here, we explored the feasibility of urinary cfDNA (ucfDNA) analysis in patients with oligometastatic colorectal cancer (CRC). Methods: We applied error-corrected targeted next-generation sequencing (NGS) (AVENIO ctDNA surveillance assay) to 10 patients with oligometastatic CRC to the liver who underwent curative-intent surgery. 40 paired samples (tumor, plasma, urine supernatant, peripheral blood mononuclear cells (PBMCs)) were collected, from which DNA was extracted. Plasma and urine cfDNA samples (acquired preoperatively) were sequenced to a median de-duplicated depth of 8,065X with on-target rate of 71%. Similarly high depth targeted NGS was applied to tumor and PBMC samples. Variants were called from plasma cfDNA and tumoral DNA and queried in ucfDNA using methods derived from CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) with integrated digital error suppression (iDES). Clonal hematopoiesis was accounted for by removing mutations also present in PBMCs. Results: Patients had a median of 2 tumors resected (range 1-7) with average sum of longest diameters of 8.7 cm (range 0.6-11). We used an average of 10 ml of blood and 37 ml of urine for cfDNA extraction. Circulating tumor DNA (ctDNA) was detected in all preoperative plasma samples (n = 10) at a mean mutant allele fraction (MAF) of 2.35% (range 0.09-11.78). Plasma ctDNA MAFs correlated significantly with tumor SLD (Pearson r = 0.81, P = 0.03). The average number of single nucleotide variants detected in plasma cfDNA, tumor and ucfDNA was 8 (range 1-42), 4 (range 1-7) and 2 (range 0-11), respectively. Indicative of concordance, 79% of the mutations called in tumor were detected in plasma. Tumor-derived DNA was detected in the preoperative urine of 6 patients (60%) with a MAF of 0.05% (range 0.03-0.12), ~35-fold less than in plasma (MAF of 1.78%, range 0.09-7.94) (P = 0.004). 38% of mutations identified in tumor and 26% from plasma were detected in ucfDNA for these 6 patients. In two patients, targetable mutations (BRAF p.Asp594Gly and PIK3CA p.Glu545Lys) present in tumor were also detected in ucfDNA, with the latter not detected in plasma. Conclusions: Our results indicate that ucfDNA analysis in patients with oligometastatic CRC is feasible; however, the mutational levels and detection sensitivity were much lower than from plasma. Yet, there may be situations where actionable mutations are missed in plasma but detected in urine.
Plasma ctDNA sequencing supplementary to tissue-based molecular profiling as a source of additional actionable information in advanced cancers.
Authors: Hancock Bradley Allen , Wong Swee Seong , Sinha Saloni , Barber Tom , Salisbury Benjamin A. , Bray Steven M. , Smallwood Lori A. , Schneider Bryan P. , Radovich Milan
Published: May 2020

e15554

Background: The standard for tumor molecular profiling in precision oncology is the tissue-based biopsy. However, tissue biopsies are limited in the ability to capture the bulk of heterogeneity in advanced cancers. The integration of liquid biopsy diagnostics complementary to tissue-based sequencing may provide a broader perspective of heterogeneity and actionability. Methods: Patients with metastatic solid tumors were evaluated in the Indiana University Health Precision Genomics (PG) Program. We retrospectively analyzed existing genomic data from matched plasma and tissue that were collected within a 90-day window. Plasma sequencing was generated with a CLIA-certified test: FoundationACT/FoundationLiquid. Tumor tissue sequencing was performed either with CLIA-grade whole exome or a gene-panel. All sequencing data was reviewed by a molecular tumor board for the determination of a personalized treatment strategy. Molecular data for this analysis was concatenated and analyzed for mutations present in ctDNA but absent in tissue. Clinical actionability was defined based on clinical evidence of a biomarker to guide therapy or match a patient to a clinical trial. Results: 288 subjects with tissue- and plasma-derived mutation data were analyzed. Discordance, defined by the presence of a somatic mutation in the plasma that was not present in the tumor tissue, was detected in 156/288 (54%) of pairs. Interestingly, the discordance was clinically actionable in 26/288 (9%) subjects. In the 32 gene-level instances of actionable discordance, the 3 top mutated genes were PIK3CA (7), PTEN (4), and EGFR (4). Discordant mutations in ATM and CHEK2, which are actionable with PARP inhibitors, but also frequently mutated in clonal hematopoiesis of indeterminant potential (CHIP) represented 4/26 (15%) instances. A complete and formal CHIP analysis is ongoing. As a result of plasma discordance, targeted interventions were employed in 3/26 (12%) subjects targeting high TMB with immunotherapy, an ERBB2 mutation with ado-trastuzumab emtansine, and a BRCA1 mutation with olaparib. All 3 patients were non-responders. Conclusions: Discordant mutations that appear in ctDNA but not in matched tumor tissue is a common occurrence. The majority of these mutations are either not druggable or variants of unknown significance, but a significant subset would be deemed clinically actionable. Clinical efficacy of targeted intervention based on actionable liquid biopsy discordance warrants further investigation.
Correlation of levels of extracellular vesicles in peripheral and pulmonary blood plasma with pathological stages of lung cancer patients.
Authors: Kim Hyun Koo , Choi Byeong Hyeon , Quan Yu Hua , Rho Jiyun , Hong Sunghoi , Park Yong , Choi Yeonho , Park Ji-Ho , Yong Hwan Seok , Han Kook Nam , Choi Young Ho
Published: May 2020

e15558

Background: Exosome concentration is known to be higher in cancer patients than in healthy individuals. In this study, we observed that the levels of exosomes differ in tumor-draining pulmonary blood and in peripheral blood in animal models and human subjects at different pathological stages of lung cancer. Methods: Ten rabbits and 40 humans formed the study cohorts. Blood was collected from a peripheral vein in all groups, and pulmonary blood was collected intraoperatively from all groups, except the healthy human controls. Quantitative analysis of exosomes was performed by nanoparticle tracking assay, CD63 enzyme-linked immunosorbent assay, and western blotting. Results: The peripheral blood of lung cancer-bearing animals and patients with lung cancer carried higher amounts of exosome than that from healthy controls ( p < 0.01 and p < 0.001, respectively). Moreover, pulmonary blood from lung cancer-bearing animals and patients had significantly higher exosome levels, compared to preoperative peripheral blood ( p < 0.01 and p < 0.0001, respectively). In patients, pulmonary exosome levels showed higher correlation with pathological stages of lung cancer than the peripheral exosome levels. Conclusions: Exosome levels increased with increasing grade of lung cancer, and this trend was more prominent in the pulmonary than in the peripheral blood.
A single institution experience with droplet digital polymerase chain reaction (dd-PCR) liquid biopsy (LB) for therapeutic decision in advanced solid tumors.
Authors: Murad Andre Marcio , Casali-da-Rocha José Claudio , Carneiro Juliana Garcia
Published: May 2020

e15562

Background: Droplet digital polymerase chain reaction (ddPCR) is a promising method for analyzing minor amounts of cell-free circulating free nucleic acid (DNA and RNA) due to its high sensitivity, low cost, and fast reading, since it dispenses bioinformatics, making it an appropriate alternative to new generation sequencing (NGS) for the detection of biomarkers to guide molecularly targeted cancer therapy. The assay covers main actionable hotspot alterations across many actionable genes: EGF(mutations), ALK (fusion, mutations), ROS1(fusion), BRAF(mutations), KRAS (mutations), NRAS(mutations), PIK3CA (mutations), ERBB2(CNV- copy number variation), ESR1(mutations), KIT(CNV) and PDGFRA(CNV). Therefore, our genes can be used in panels, both in therapeutically applied genotyping and in detecting molecular responses as well as in secondary resistance to tumors such as NSCLC, breast, cervix. rectal, GIST, melanoma and pancreas. Methods: dd-PCR was performed using the QX200 system (BIO-RAD, Hercules). The extraction of DNA and RNA was done using the magnetic beads technique of Thermofisher's MagMAX Kit. All samples were tested in duplicate. Up to now, 54 metastatic cancer patients were tested: NSCLC - 14 (26%), breast - 11 (20%), colorectal - 11 (20%), melanoma - 9(17%), pancreas - 4(7%), ovarian - 3(6%) and salivary glands - 2 (4%). Results: Significant genomic alterations were detected in 19 (35%) patients: 5 (9%) mutations in the KRAS G12V gene (all in colorectal cancer), 5 (9%) ERBB2 amplification (breast cancer), 2 (3.65%) EGFR L858R mutations (NSCLC), 2 (3.5%) EGFR del19 mutations (NSLC), 2 (3.5%) EGFR T790M mutations (NSCLC), 2 (3.5%) BRAF-V600E mutations (colon and melanoma), 1 (1.8%) ALK-EML4 fusion (NSCLC). The MAF (Mutant Allele Fraction) ranged from 0.9% to 24%. In all cases, the results were decisive for the indication or change of a targeted therapy. Median turnaround time was 36 hours and the average cost of the panels was around 500 USD (median of 4 genes per panel). Conclusions: Our results suggest that dd-PCR is a highly sensitive method and could be used for a routine laboratory detection of the important genomic variations to determine the targeted therapy in patients with varied advanced solid tumors.