Large biological datasets are being produced at a rapid pace and create substantial storage challenges, particularly in the domain of high-throughput sequencing (HTS). Most approaches currently used ...to store HTS data are either unable to quickly adapt to the requirements of new sequencing or analysis methods (because they do not support schema evolution), or fail to provide state of the art compression of the datasets. We have devised new approaches to store HTS data that support seamless data schema evolution and compress datasets substantially better than existing approaches. Building on these new approaches, we discuss and demonstrate how a multi-tier data organization can dramatically reduce the storage, computational and network burden of collecting, analyzing, and archiving large sequencing datasets. For instance, we show that spliced RNA-Seq alignments can be stored in less than 4% the size of a BAM file with perfect data fidelity. Compared to the previous compression state of the art, these methods reduce dataset size more than 40% when storing exome, gene expression or DNA methylation datasets. The approaches have been integrated in a comprehensive suite of software tools (http://goby.campagnelab.org) that support common analyses for a range of high-throughput sequencing assays.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We present GobyWeb, a web-based system that facilitates the management and analysis of high-throughput sequencing (HTS) projects. The software provides integrated support for a broad set of HTS ...analyses and offers a simple plugin extension mechanism. Analyses currently supported include quantification of gene expression for messenger and small RNA sequencing, estimation of DNA methylation (i.e., reduced bisulfite sequencing and whole genome methyl-seq), or the detection of pathogens in sequenced data. In contrast to previous analysis pipelines developed for analysis of HTS data, GobyWeb requires significantly less storage space, runs analyses efficiently on a parallel grid, scales gracefully to process tens or hundreds of multi-gigabyte samples, yet can be used effectively by researchers who are comfortable using a web browser. We conducted performance evaluations of the software and found it to either outperform or have similar performance to analysis programs developed for specialized analyses of HTS data. We found that most biologists who took a one-hour GobyWeb training session were readily able to analyze RNA-Seq data with state of the art analysis tools. GobyWeb can be obtained at http://gobyweb.campagnelab.org and is freely available for non-commercial use. GobyWeb plugins are distributed in source code and licensed under the open source LGPL3 license to facilitate code inspection, reuse and independent extensions http://github.com/CampagneLaboratory/gobyweb2-plugins.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
High-throughput data can be used in conjunction with clinical information to develop predictive models. Automating the process of developing, evaluating and testing such predictive models on ...different datasets would minimize operator errors and facilitate the comparison of different modeling approaches on the same dataset. Complete automation would also yield unambiguous documentation of the process followed to develop each model. We present the BDVal suite of programs that fully automate the construction of predictive classification models from high-throughput data and generate detailed reports about the model construction process. We have used BDVal to construct models from microarray and proteomics data, as well as from DNA-methylation datasets. The programs are designed for scalability and support the construction of thousands of alternative models from a given dataset and prediction task. Availability and Implementation: The BDVal programs are implemented in Java, provided under the GNU General Public License and freely available at http://bdval.campagnelab.org Contact: fac2003@med.cornell.edu
BackgroundThe immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and antitumor activity has posed a major challenge for developing effective immunotherapies ...for solid tumors. Chimeric antigen receptor (CAR)-engineered T cell therapy has shown unprecedented clinical response in treating patients with hematological malignancies, and intense investigation is underway to achieve similar responses with solid tumors. Immunologically cold tumors, including prostate cancers, are often infiltrated with abundant tumor-associated macrophages (TAMs), and infiltration of CD163+ M2 macrophages correlates with tumor progression and poor responses to immunotherapy. However, the impact of TAMs on CAR T cell activity alone and in combination with TME immunomodulators is unclear.MethodsTo model this in vitro, we utilized a novel co-culture system with tumor cells, CAR T cells, and polarized M1 or M2 macrophages from CD14+ peripheral blood mononuclear cells collected from healthy human donors. Tumor cell killing, T cell activation and proliferation, and macrophage phenotypes were evaluated by flow cytometry, cytokine production, RNA sequencing, and functional blockade of signaling pathways using antibodies and small molecule inhibitors. We also evaluated the TME in humanized mice following CAR T cell therapy for validation of our in vitro findings.ResultsWe observed inhibition of CAR T cell activity with the presence of M2 macrophages, but not M1 macrophages, coinciding with a robust induction of programmed death ligand-1 (PD-L1) in M2 macrophages. We observed similar PD-L1 expression in TAMs following CAR T cell therapy in the TME of humanized mice. PD-L1, but not programmed cell death protein-1, blockade in combination with CAR T cell therapy altered phenotypes to more M1-like subsets and led to loss of CD163+ M2 macrophages via interferon-γ signaling, resulting in improved antitumor activity of CAR T cells.ConclusionThis study reveals an alternative mechanism by which the combination of CAR T cells and immune checkpoint blockade modulates the immune landscape of solid tumors to enhance therapeutic efficacy of CAR T cells.
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Background: Serum-based tumor markers (STM) (AFP, LDH, and b-hCG) used as standard of care in patients with testicular cancer lack sufficient sensitivity and specificity for molecular residual ...disease (MRD) detection. Although circulating tumor (ct) DNA holds promise as a prognostic biomarker in a variety of malignancies, its clinical utility in testicular cancer has not been well characterized. This study evaluates the utility of longitudinal ctDNA monitoring for MRD detection in patients with testicular cancer. Methods: Retrospective ctDNA analysis was performed using a personalized, tumor-informed ctDNA assay (SignateraTM bespoke mPCR-NGS assay). ctDNA was evaluated during the MRD 1-12 weeks post-orchiectomy and surveillance >12 weeks post-orchiectomy, post-adjuvant chemotherapy (ACT), or after retroperitoneal lymph node dissection (RPLND) windows. Correlation between ctDNA status and patient outcomes (event-free survival (EFS)) was assessed. EFS is described as the interval from radical orchiectomy to the date of radiological recurrence or any evidence of residual/persistent disease after the completion of ACT or RPLND. A clinical nomogram was developed to predict clinical outcomes based on age, stage, histology, presence of STMs, and ctDNA during the surveillance window. This point system calculates the 1 2-year EFS probability with patients receiving points between 0-200. Results: A total of 145 plasma samples were collected from 35 patients with stages I-III testicular cancer (%stage I/II/III: 66/23/11; %seminoma/non-seminoma: 49/51). Post-operatively, 43% (15/35) of these were on surveillance, 23% (8/35) received ACT, 8.6% (3/35) underwent RPLND, and 26% (9/35) underwent RPLND and received ACT. The median age of the cohort was 34 years (IQR: 29-42) with a median follow-up of 10 months (IQR: 6.5-18.7).Pre-orchiectomy (N=15), ctDNA was detected in 91.6% (11/12) of stage I and 100% of stage II/III (3/3)) patients.During the MRD (N=22) and surveillance (N=27) windows, patients who tested ctDNA positive showed a significantly inferior EFS compared to those who tested negative (MRD: HR 7.2, 95% CI: 1.4-36.7, p=0.017 and Surveillance: HR 11.8, 95% CI: 2.3-59.1, p=0.003). Multivariate regression analysis during surveillance revealed any-time ctDNA-positivity as the only factor significantly associated with poor EFS (p=0.015) when compared to other clinicopathological features such as age, stage, histology, and elevated STMs. Conclusions: This is the first study that utilizes longitudinal tumor-informed ctDNA testing to assess patient outcomes and disease status. Our results suggest that tumor-informed ctDNA analysis may hold promise as a biomarker for EFS in patients with testicular cancer. As such, we developed the first-ever clinical nomogram that includes ctDNA status and other clinicopathological factors to stratify patient outcomes.
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Background: Transurethral resection of bladder tumor (TURBT) plus systemic therapy has been known for decades to achieve durable bladder-intact survival in a subset of patients with MIBC but ...efforts to advance this paradigm have been complicated by a lack of (a) prospective studies, (b) rigorous approaches to assess and define clinical complete response (cCR), and (c) integration of novel therapies. Methods: Eligible patients were cisplatin-eligible with cT2-T4aN0M0 urothelial bladder cancer. Patients received 4 cycles of gemcitabine, cisplatin, plus nivolumab followed by clinical restaging including urine cytology, MRI/CT of the bladder, cystoscopy and bladder biopsies. Patients achieving a cCR (normal cytology, imaging, and cT0/Ta) were eligible to proceed without cystectomy and receive nivolumab q2 weeks x 8 followed by surveillance. Patients not achieving cCR were recommended to undergo cystectomy. Coprimary endpoints included (1) cCR rate and (2) association between cCR and 2-year outcomes. The key secondary endpoint was the impact of pre-specified baseline genomic alterations on outcomes. Additional biomarkers to refine patient selection were also explored. Results: Between 8/2018-11/2020, 76 patients were enrolled at 7 sites (male 79%, median age 69; cT2 = 56%, cT3 = 32%, cT4 = 12%). Median follow-up is 27 months. 72/76 patients underwent clinical restaging and a cCR was achieved in 33/76 (43%; 95% CI: 32%, 55%). One cCR patient opted for immediate cystectomy (ypTaN0M0). Outcomes are summarized in the Table. Baseline ERCC2, ATM, FANCC, or RB1 alterations were not, but tumor mutational burden ≥ 10 mutations/mb was, significantly associated with the composite endpoint of ypT0 (immediate cystectomy) or 2-year bladder-intact metastasis-free survival (BIMFS). On landmark analysis, VI-RADS (Vesical Imaging–Reporting and Data System) score (3-5 versus 1-2) on restaging MRI (central blinded review) was associated with inferior BIMFS (HR 4.5; p = <0.01) and MFS (HR 19.3; p <0.01). Circulating tumor DNA data will be presented at the meeting. Conclusions: TURBT followed by gemcitabine, cisplatin, plus nivolumab achieves stringently defined cCR in a substantial subset of patients with MIBC. ≥2-year bladder-intact survival is achieved in the majority of patients with a cCR. Clinical trial information: NCT03558087 . Table: see text
5034 Background: Serum tumor markers (STM) (AFP, hCG) are currently utilized in the management of patients (pts) with testicular cancer. However, in a substantial proportion of pts STM can be normal ...or falsely elevated. We evaluated the clinical utility of longitudinal ctDNA monitoring as a prognostic marker in pts with testicular cancer. Methods: Longitudinal analysis was performed on a multi-institutional cohort of pts with stages I-III testicular cancer using a clinically validated, personalized, tumor-informed 16-plex PCR ctDNA assay (Signatera, Natera Inc.). ctDNA was evaluated pre-orchiectomy, during the MRD (1-12 weeks post-orchiectomy) and surveillance windows (>12 weeks post-orchiectomy, after retroperitoneal lymph node dissection RPLND, and/or chemotherapy). The correlation between ctDNA status and event-free survival (EFS) was assessed. EFS is described as the interval from orchiectomy to the date of radiological recurrence or any evidence of residual/persistent disease post-completion of chemotherapy or RPLND. This analysis includes 28 pts from Icahn School of Medicine, 20 pts from Indiana University, and 7 pts from City of Hope. Results: Plasma samples (n=197) were collected from 55 pts - %stage I/II/III: 42/22/36; %seminoma/non-seminoma: 31/69. The median age was 34 years (range: 16-67), and the median follow-up was 11 months (range: 2-76). Disease management post-orchiectomy included surveillance in 27% (15/55), RPLND in 11% (6/55), chemotherapy in 40% (22/55), and chemotherapy+RPLND in 22% (12/55) of the pts. Pre-orchiectomy ctDNA was detectable in 14/15 pts - 91.6% (11/12) of pts with stage I, and 100% (3/3) of pts with stage II/III disease. ctDNA evaluation pre-RPLND (N=7; 1 seminoma, 6 non-seminoma) revealed ctDNA-positivity in 6 pts. Six pts had ctDNA testing performed pre- and post-completion of chemotherapy for stage II/III disease; ctDNA exhibited a median MTM/mL reduction of 96.5% (range: 75.6–100) post-treatment completion. Seventeen pts had ctDNA testing post-RPLND or chemotherapy; 0/9 pts with undetectable ctDNA relapsed while 4/8 pts with detectable ctDNA experienced clinical recurrence at the most recent evaluation with follow-up ongoing. During the MRD (N=27) and surveillance (N=36) windows, pts with detectable vs. undetectable ctDNA showed a significantly inferior EFS (MRD: HR= 5.27, 95% CI: 1.22-22.71; p=0.026. Surveillance: HR= 10.8, 95% CI: 2.53-46.01; p=0.001). During the surveillance window, elevated STM vs. normal STM was not associated significantly with worse EFS (HR= 2.01, 95%Cl: 0.71-5.72; p=0.191). Conclusions: Tumor-informed ctDNA analysis shows promise for MRD detection in pts with testicular cancer. With further study, ctDNA monitoring may have utility in clinical decision-making. Larger prospective trials are planned for validation of clinical utility.
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Background: Transurethral resection of bladder tumor (TURBT) plus systemic therapy has been known for decades to achieve durable bladder-intact survival in a subset of patients ...with MIBC but efforts to advance this paradigm have been complicated by (a) lack of prospective studies exclusively testing cisplatin-based neoadjuvant chemotherapy, (b) lack of rigorous methods to define clinical complete response (cCR) and its association with long term outcomes and (c) limited understanding of the role of “salvage” cystectomy. Methods: Eligible patients were cisplatin-eligible with cT2-T4aN0M0 urothelial bladder cancer. Patients received 4 cycles of gemcitabine, cisplatin, plus nivolumab followed by clinical restaging including urine cytology, MRI/CT of the bladder, cystoscopy and bladder/prostatic urethral biopsies. Patients achieving a cCR (normal cytology, imaging, and cT0/Ta) were eligible to proceed without cystectomy and receive nivolumab q2 weeks x 8 followed by surveillance; otherwise, patients underwent cystectomy. Coprimary endpoints included (1) cCR rate and (2) ability of cCR to predict 2-year metastasis-free survival (MFS). The key secondary endpoint was the impact of genomic alterations in baseline TURBT (TMB, ERCC2, FANCC, RB1, ATM) on performance of cCR for predicting MFS. The cCR rate coprimary endpoint, and interim analysis of 1-year outcomes, are reported. Results: Between 8/2018-11/2020, 76 patients were enrolled at 7 sites (male 79%, median age 69; cT2 = 56%, cT3 = 32%, cT4 = 12%) and 64 (84%) have completed post-cycle 4 restaging; 31/64 achieved a cCR (48%; 95% CI 36%, 61%). The median follow-up of cCR patients is 13.7 months (range, 2.5-24 months). One cCR patient opted for immediate cystectomy (pTaN0M0). Outcomes for the entire cohort are summarized in the table below. Local recurrence has occurred in 8/31 cCR patients and 6 underwent cystectomy (pT0N0 = 1, pTaN0 = 1, pTisN0 =1, pT2N0 = 2, pT4N1 = 1). TMB ≥ 10 mut/Mb (p=0.02) or mutant ERCC2 (p=0.02) were associated with cCR or pT0. Conclusions: TURBT + gemcitabine, cisplatin, plus nivolumab achieves stringently defined cCR in a large subset of patients with MIBC. 1-year bladder intact survival is possible though the durability of responses, and role of genomic biomarkers in management algorithms, requires longer follow-up. Clinical trial information: NCT03558087. Table: see text
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Background: Cabozantinib (C) may enhance response to immune checkpoint inhibitors (ICIs) by promoting an immune-permissive microenvironment and has shown encouraging activity in ...combination with ICIs in tumor types including RCC and HCC. C and atezolizumab (A) have shown low objective response rates as monotherapy in metastatic castration-resistant prostate cancer (mCRPC) (Smith JCO 2012; Kim JCO 2018). COSMIC-021 (NCT03170960), a multinational phase 1b study, is evaluating the combination of C + A in various solid tumors. We report results for Cohort 6 in mCRPC. Methods: Eligible patients (pts) were required to have radiographic progression in soft tissue after enzalutamide and/or abiraterone, measurable disease, and an ECOG PS of 0 or 1. Prior chemotherapy for mCSPC was permitted. Pts received C 40 mg PO QD and A 1200 mg IV Q3W. CT/MRI scans were performed Q6W for the first year and Q12W thereafter. The primary endpoint is ORR per RECIST 1.1. Other endpoints include safety, ORR per irRECIST, duration of response (DOR), PFS, and OS. Results are presented for the first 44 pts enrolled. Results: Median follow-up as of Dec 20, 2019 was 12.6 mo (range 5, 20) for the 44 mCRPC pts. Median age was 70 y (range 49, 90), 50% had ECOG PS 1, 34% had visceral metastases, and 61% had extrapelvic lymph node metastases. 27% had prior docetaxel and 52% had 2 prior novel hormonal therapies. The most common any grade treatment-related adverse events (TRAEs) were fatigue (50%), nausea (43%), decreased appetite (39%), diarrhea (39%), dysgeusia (34%), and PPE (32%). One grade 5 TRAE of dehydration was reported in a 90 y/o. Median duration of treatment was 6.3 mo. ORR per RECIST 1.1 among all 44 pts was 32% (2 CRs 4.5% and 12 PRs 27%); 21 (48%) pts had SD resulting in a disease control rate of 80% in all pts. One pt with PD per RECIST 1.1 had an irPR per irRECIST. ORR per RECIST 1.1 was 33% in 36 pts with high-risk disease (visceral and/or extrapelvic lymph node metastases). Median DOR for all pts with response per RECIST 1.1 was 8.3 mo (range 2.8, 9.8+). 17 (50%) of 34 pts with post-baseline PSA evaluation had a decrease in PSA. In 12 responders with post-baseline PSA evaluation, 8 (67%) had a PSA decrease ≥50%. Tumor PD-L1 expression will also be reported. Conclusions: The combination of C + A had a tolerable safety profile and demonstrated clinically meaningful activity with durable responses in men with mCRPC. Given the encouraging activity in these pts, especially in those with high-risk disease, further evaluation of C + A in men with mCRPC is being pursued. Clinical trial information: NCT03170960 .
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Background: C may enhance response to immune checkpoint inhibitors by promoting an immune-permissive microenvironment. COSMIC-021 (NCT03170960), a multinational phase 1b study, is ...evaluating the combination of C with A in various solid tumors. We report interim results from Cohort 6 in mCRPC. Methods: Eligible pts were required to have radiographic progression in soft tissue after enzalutamide and/or abiraterone, measurable disease, and an ECOG PS of 0 or 1. Prior chemotherapy for mCSPC was permitted. Pts received C 40 mg PO qd and A 1200 mg IV q3w. CT/MRI scans were performed q6w for 52w and q12w thereafter. Bone scans were performed q12w. The primary endpoint is ORR per RECIST 1.1. Other endpoints include safety, ORR per irRECIST, duration of response (DOR), PFS, and OS. Results: As of Oct 2019, 44 mCRPC pts were enrolled with a median follow-up of 10.6 mo (range 3.4+, 17.9). Median age was 70 y (range 49, 90), 50% had ECOG PS 1, 34% had visceral metastases, and 61% had extrapelvic lymph node metastases. 27% of pts had prior docetaxel and 52% had ≥2 prior novel hormonal therapies. The most common any grade TEAEs were fatigue (57%), nausea (48%), decreased appetite (45%), diarrhea (39%), PPE (32%), and vomiting (32%). One Grade 5 TRAE of dehydration was reported in a 90 y/o. Median duration of treatment was 5.3 mo. The ORR per RECIST 1.1 among all pts was 32% (2 CRs 4.5% and 12 PRs 27%); 21 (48%) pts had SD giving a disease control rate of 80% in all pts. One pt with initial PD per RECIST 1.1 had an irPR per irRECIST. ORR per RECIST 1.1 was 33% in 36 pts with high-risk clinical features (visceral and/or extrapelvic lymph node metastases). Median DOR for all pts with response per RECIST 1.1 was 8.3 mo (range 1.38+, 9.76+). In 12 responders with at least 1 post-baseline PSA evaluation, 8 (67%) had a PSA decline ≥50%. Conclusions: The combination of C+A had a tolerable safety profile and demonstrated clinically meaningful activity with durable responses in men with mCRPC. Given the encouraging activity in these pts, especially in those with visceral and/or extra pelvic lymph node metastases, further evaluation of C+A in men with mCRPC is being pursued. Clinical trial information: NCT03170960.
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