IntroductionLarge B-cell lymphoma (LBCL) taxonomy has moved in the direction of a molecular classification, but further clinical experience is needed. We present high-risk gene mutations, which predict outcome in an exploratory study of a consecutive real-world cohort of patients with primary LBCL treated with R-CHOP or R-CHOP-like therapy.MethodsThe study was a Registry Study Research Project. Sixty-one patients with LBCL, who had a diagnostic tumor sample successfully examined with a 59-gene next-generation sequencing (NGS) panel as a part of routine clinical work, were included in an otherwise unselected cohort. Data were extracted from patient files and pathology reports.ResultsMutations in NOTCH2 (HR 9.69; 95% CI [2.46-38.11]; p = 0.0012), PRDM1 (HR 3.54; 95% CI [1.03-12.22]; p = 0.045), and TP53 (HR 5.89; 95% CI [1.71-20.32]; p = 0.005) were significantly associated with inferior survival in patients with primary LBCL treated with intention to cure with at least three series of R-CHOP or R-CHOP-like therapy. Neither MYD88 (HR 0.66; 95% CI (0.17-2.49), p = 0.54) nor CD79B (HR 0.84;95% CI (0.18-3.88), p = 0.82) mutations were associated with inferior survival.ConclusionWith a targeted gene panel and NGS methodology feasible in daily diagnostic routine, we identified high-risk gene mutations with a significant prognostic impact of which TP53 mutations were reproducible across validation cohorts.

Marginal zone lymphoma (MZL) is an uncommon non-Hodgkin B-cell lymphoma accounting for 7-10% of lymphoma diagnoses. Extranodal MZL (EMZL) of mucosa-associated lymphoid tissue (MALT) is the most common subtype (61%) of MZL. While the mutational landscape, chromosomal aberrations and transcriptome of nodal and splenic MZL have been extensively evaluated, the mutational landscape of EMZL has only been evaluated in a small number of patients and mainly by targeted sequencing. Consequently, the comprehensive genomic landscape of EMZL remains largely uncharacterized. Herein, we performed whole exome sequencing followed by targeted sequencing of potential tumor driver genes, copy number and/or RNA transcriptome analyses in 165 patients with pretreatment EMZL tumors involving 16 anatomic locations. All samples were collected and underwent expert review as part of the Atlas of Blood Cancer Genomes consortium. The most common anatomic locations were ocular adnexa (OAMZL) (n=35), gastric (n=25, including 6 positive for Helicobacter pylori), salivary glands (n=25), and lungs (n=24). Using common criteria for variant mutation calling and significant focal copy number (CN), we identified 1218 variants (1030 missense and 188 truncation mutations) across 174 genes and 2837 CN focal alterations (1614 copy gains and 1223 copy number losses). Of these genes, 44 and 11 were mutated in >5% and >10% of specimens, respectively. We detected variants identified previously by targeted sequencing in EMZL from different locations (e.g. TNFAIP3 (A20) (12%), TBL1XR1 (13%), SPEN (10%), CARD11 (7%), TET2 (7%) and CREBBP (6%)). We detected mutations in KLF2 (10 mutations in 8 EMZL patients) and PTPRD (7 mutations in 7 EMZL patients) that were previously suggested to be specific for splenic and nodal MZL, respectively. We also detected variants not previously reported in EMZL, including a transcriptional factor ZEB2, LRP1B,and others. The most frequently mutated gene in our study (25% of tumors) was IGLL5. Some mutations were limited to specific locations (e.g. ZEB2 mutations seen exclusively in gastric and salivary gland EMZL), while most were observed across all anatomic locations. There was no association between specific mutations and clonal IGHV, in contrast to previous reports. Using Enrichr tool, we observed enrichment of mutations in genes belonging to the B cell receptor activation, NOTCH signaling, Wnt-beta catenin signaling, NF-kB signaling , PI3K AKT signaling, DNA repair and other pathways. Chromosomal and copy number changes were also commonly observed including previously reported trisomy of chromosomes 3 and 18. Combining DNA mutations and CN alterations, the number of genetic lesions per tumor sample in the 174 mutated genes ranged from 0 to 95, with an average load of 17 lesions per case. RNA sequencing was successful in 159 EMZL tumors from 16 distinct anatomic locations. Unsupervised clustering using all genes with a median log2-normalized gene expression value greater than 5 and a standard deviation greater than 1 (1323 genes) demonstrated that the majority of gastric EMZL with and without H. pylori infections clustered together on 2 dendrograms as did most of the samples of OAMZL. Since the clustering could be forced by organ-specific and not tumor-specific gene expression, we next focused on the expression of the 345 genes from the LM22 matrix that passed our transcriptomic row filtering. Unsupervised clustering resulted in clustering of most gastric EMZL irrespective of H. pylori positivity together in the same dendrogram branch, accompanied by few additional EMZL from other anatomic locations. Most of the EMZL samples from other locations did not show preferential co-clustering together. On comparison of gene expression between gastric and OAMZL, there was enrichment of the B-cell receptor signaling pathway, B-cell activation, B-cell naïve and B-cell memory cells, B-cell proliferation and NF-kB signaling in the latter. Immune microenvironment analysis showed a statistically significant increase in resting mast cells and decrease in M1 macrophages in gastric EMZL and increase in regulatory T cells in salivary EMZL. The broad genomic studies reported here underscore the biological similarity of EMZL across anatomical sites with the potential exceptions of gastric EMZL (regardless of H. Pylori status). These data provide valuable genomic and transcriptomic resources to inform future diagnostic and therapeutic strategies.

Next-generation sequencing (NGS) affords comprehensive insights into the genomic landscape of lymphomas. We examined the mutational pattern in patients with Waldenström macroglobulinemia (WM) or lymphoplasmacytic lymphoma (LPL) as well as the diagnostic and clinical utility of a tailored NGS lymphoma panel. A consecutive series of 45 patients was reviewed and NGS analysis was performed as part of a routine diagnostic setup. The custom designed NGS panel assayed all coding sequences of 59 genes of known clinical significance in lymphoid neoplasms. The most frequently mutated genes were MYD88, CXCR4, BIRC3, CD79B, and ARID1A. Additional somatic mutations were detected in 17 genes with four mutations categorized as pathogenic or likely pathogenic. BIRC3 and TP53 mutations were associated with adverse clinical phenotypes. NGS performance for the MYD88L265P variant was 96% when compared to qPCR. In conclusion, targeted NGS provided important diagnostic and prognostic information in a routine clinical setting.

INTRODUCTION:Large B-cell lymphoma (LBCL) exhibits striking clinical and molecular heterogeneity. New approaches have emerged to explore tumor heterogeneity and classify LBCL into biological categories. Consequently, the informational requirements from diagnostic samples to provide the necessary information have increased, but the adequacy of single-site biopsies to provide such information is largely unknown. Here we describe spatial and temporal intra-patient variations in the mutational landscape of paired biopsies. METHODS:Paired biopsies from 30 patients with LBCL were obtained from spatially distinct sites at the time of primary diagnosis before treatment and/or at a subsequent relapse. The samples were sequenced using a custom designed 59-gene next generation sequencing (NGS) lymphoma panel. RESULTS:Differences in detected mutations of pathogenic or likely pathogenic significance were frequent both when comparing paired diagnostic biopsies, 2/6 (33%), and when comparing paired biopsies at primary diagnosis and relapse, 8/16 (50%). Mutational heterogeneity tended to increase with longer time interval between biopsies. Analysis of paired diagnostic and relapse biopsies revealed that certain clones present at diagnosis disappeared, while new clones emerged at relapse. Notably, TP53 mutations were detected in six out of seven patients in an extranodal location. In two cases, TP53 mutation was only detected in the relapse biopsy. Several of the mutations identified in this study are used or under investigation as targets for cancer treatments. CONCLUSION:Multi-site biopsies revealed spatial and temporal mutational heterogeneity in patients with LBCL. Our findings indicate that mutational differences between biopsy pairs can occur at all timepoints examined. This underscores the necessity of performing repeat biopsies with each relapse to capture the full spectrum of genetic aberrations.

Clonal hematopoiesis is highly prevalent in elderly patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), and around 20% of BPDCN patients have an associated myeloid malignancy. We present a patient with localized BPDCN and concomitant myelodysplastic syndrome (MDS), previously followed for several years due to clonal cytopenia of unknown significance. Compared targeted next-generation sequencing (NGS) of the skin tumor and sequential bone marrow samples showed shared variants in ASXL1 and TET2 with a de novo NRAS variant in both BPDCN and MDS compared to preceding bone marrow samples. These findings illustrate a shared clonal origin of BPDCN and MDS resulting from progressive clonal hematopoiesis.

INTRODUCTION:Integration of molecular characterization of lymphomas in clinical diagnostics may improve subclassification and risk-stratification, and we implemented a next generation sequencing (NGS) analysis as part of routine diagnostic work-up of all mature B-cell non-Hodgkin's lymphoma (B-NHL). Here, we present data of mutational profiles with potential complementary diagnostic, prognostic, and predictive value detected in our consecutive non-selected cohort of B-NHL patients.METHODS:NGS results from 298 patients with both newly diagnosed and relapsed/refractory disease were included as a single center study. NGS was performed as routine analysis together with standard diagnostic work-up using a custom-made amplicon PCR-based multiplex NGS panel covering all coding exons and consensus splice sites in 59 genes.RESULTS:Mutations were detected in 94% of the 298 samples. Most lymphomas could be classified definitively, but 24 cases were classified as small B-cell lymphomas without defining characteristics. Of these, 50% (12/24 cases) could retrospectively be assigned a likely diagnostic subtype according to mutational findings.CONCLUSION:Implementation of a 59 gene exome sequencing panel added diagnostic value to 50% of unclassified cases and provided in 94% of the cases possible biomarkers for disease monitoring as well as potential diagnostic, prognostic, and predictive markers for future studies.

Introduction: Precision medicine requires equivalently precise diagnostic methods. The lymphoma diagnoses are primarily based on characteristic patterns of morphology and protein expression detected by immunohistochemistry (IHC). However, integral molecular characterization of lymphomas in clinical diagnostics may improve subclassification and risk-stratification. To accommodate this growing need we implemented a next generation sequencing (NGS) analysis as part of routine diagnostic work up of all mature non-Hodgkin lymphoma (NHL). We present data of mutational profiles with potential complementary diagnostic, prognostic and predictive value detected in our consecutive non-selected cohort of NHL patients. Methods: NGS results from 320 individual consecutive non-selected diagnostic patient samples with a mature NHL were included as a single center study. Patients with both newly diagnosed and relapsed/refractory or progressive disease were included. Diagnoses were according to WHO (4.rev edition). NGS was performed as routine analysis together with standard diagnostic work-up using a custom-made amplicon PCR-based multiplex NGS-panel covering all coding exons and consensus splice sites in 59 genes. Results: Patients are presented in Table 1. Mutations were detected in 93% of the 320 samples. Most B-cell lymphomas (BCL) could be classified definitively and had characteristic mutational profiles as shown in Figure 1, but 24 cases were classified as small cell B-cell lymphomas without defining characteristics (SBCL-NOS). 50% (12/24 cases) of SBCL-NOS could retrospectively be assigned a likely diagnostic subtype according to mutational findings. (1 FL, 6 MZL, 3 LPL and 2 Small lymphocytic lymphoma). Conclusion: Implementation of NGS in routine diagnostics of mature B-cell NHL added diagnostic value to 50% of unclassified cases and provided in a total of 93% of all cases possible biomarkers for disease monitoring as well as potential diagnostic, prognostic and predictive markers for future studies. The research was funded by: Department of Pathology, Herlev-Gentofte Hospital, Denmark Keywords: Diagnostic and Prognostic Biomarkers, Pathology and Classification of Lymphomas No conflicts of interests pertinent to the abstract.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, both regarding clinical presentation, response to treatment and outcome. Recently, subclassification of DLBCL based on mutational profile has been suggested, and next generation sequencing (NGS) analysis may be relevant as part of the diagnostic workflow. This will, however, often be based on analysis of one tumor biopsy. Here, we present a prospective study where multi-site sampling was performed prior to treatment in patients with newly diagnosed DLBCL. Two spatially different biopsies from 16 patients were analyzed using NGS with an in-house 59-gene lymphoma panel. In 8/16 (50%) patients, mutational differences were found between the two biopsy sites, including differences in TP53 mutational status. Our data indicate that a biopsy from the extra-nodal site may represent the most advanced clone, and an extra-nodal biopsy should be preferred for analysis, if safely accessible. This will help ensure a standardized stratification and treatment decision.

Previous studies indicated that compounds termed Disulfide bond Disrupting Agents (DDAs) exhibit anti-cancer activity that is associated with downregulation of EGFR/HER1, HER2, and HER3, and activation of Death Receptors 4 and 5 (DR4/5). DDA-induced HER1-3 downregulation is preceded by disulfide-mediated oligomerization. In contrast, DDA-mediated DR4/5 oligomerization results in DR5 upregulation, and activation of DR4/5 pro-apoptotic signaling through Caspases 8 and 3. However, the precise mechanisms by which altered disulfide bonding stabilizes and activates DR5 are unknown. A recent report indicated that the extracellular domain of DR5 acts in an auto-inhibitory manner to prevent DR5 oligomerization and pro-apoptotic signaling in the absence of its ligand, TRAIL. A subsequent paper showed that the DR5 auto-inhibitory domain is a positive patch consisting of three basic residues. Importantly, the structure of the auto-inhibitory loop is formed by two disulfide bonds. We hypothesize that DDAs disrupt the disulfide bonds that make up the auto-inhibitory loop, resulting in DR5 oligomerization, and activation of Caspase 8/3-driven apoptosis in a TRAIL-independent manner. Due to their novel mechanisms of action, DDAs may overcome the pharmacological liabilities that have limited the efficacy of TRAIL analogs and DR5 agonist antibodies. Another unanswered question is how precisely DDAs alter DR5 and EGFR disulfide bonding. The direct targets of DDA action were revealed through affinity purification studies with biotinylated-DDA analogs. These studies identified the protein disulfide isomerases AGR2, ERp44, and PDIA1 as DDA target proteins, explaining how DDAs alter DR5 and EGFR disulfide bonding patterns. Consistent with this interpretation, knockdown of AGR2 or ERp44, or expression of catalytically null AGR2 or ERp44 mutants, mimicked DDAs in inducing disulfide-mediated DR5 oligomerization and Caspase 8 activation. Together, these results demonstrate a fundamentally novel, ligand-independent mechanism for activation of DR5 through DDA-mediated inhibition of the PDIs AGR2, ERp44, and PDIA1. Significantly, DDAs are the first identified active site inhibitors of AGR2 and ERp44. Citation Format: Brian K. Law, Mary E. Law, Elham Yaaghubi, Amanda Ghilardi, Brad J. Davis, Renan Ferreira, Samantha Eggleston, Jade Nguyen, Grace Alexandrow, Jin Koh, Sixue Chen, Chi-Wu Chiang, Coy Heldermon, Peter Norgaard, Ronald K. Castallano, Zaafir M. Dulloo. Disulfide isomerases AGR2, ERp44, and PDIA1 maintain death receptor 5 in an auto-inhibited, monomeric form. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6148.

Background: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare B-cell malignancy characterized by infrequent neoplastic cells embedded in an immunologically active tumor microenvironment (TME). Histologically, NLPHL can be divided into classical (Fan A-B) and variant (Fan C-F) growth patterns. Clinical course is generally indolent, and the majority of patients present with limited stage disease. However, advanced stage with splenic involvement and variant histology are known to be associated with more aggressive disease. The molecular features underlying this association remain unknown. To gain further insights into disease biology, we recruited NLPHL cases as part of the Atlas of Blood Cancer Genomes (ABCG) initiative, a consortium consisting of 26 institutions. Design: We collected comprehensive clinicopathological data including splenic involvement from 135 NLPHL patients, with centralized review performed by a panel of dedicated hematopathologists to ensure accurate diagnosis per 2016 WHO criteria. We performed RNA sequencing on formalin-fixed paraffin-embedded (FFPE) diagnostic tumor lymph nodes (n=100) and enumerated tumor-infiltrating immune cell compositions using FARDEEP with signature matrix LM22 from CIBERSORT. In addition, we performed gene set enrichment analysis to compare molecular pathways between the patients with and without splenic involvement. The Kaplan-Meier method was used to calculate overall survival (OS) and progression free survival (PFS) probabilities, and the log-rank test was used for comparisons between groups. Continuous variables were compared using the Wilcoxon rank-sum test. Results: The median age at diagnosis was 42 years (range 5-80 years). Most patients were males (n=96; 71%) and had limited stage disease (n=96, 74%). Splenic involvement was present in 17 patients (13%). Histologic growth pattern was available for 97 cases, 41 (42%) with variant and 56 (58%) with classical histology. Variant histology was enriched in the patients with splenic involvement. Thirty-five (28%) relapses and 5 (3.9%) transformations occurred during a median follow-up of 80 months (range 0.36-604 months). Ten-year OS and PFS rates for the whole cohort were 86% (95% CI, 78-95%) and 59% (95% CI, 50-71%), respectively. Patients with splenic involvement at diagnosis tended to have inferior outcome at 10 years compared with those without splenic involvement (PFS 40% vs 62%, P=0.129; OS 79% vs 88%, P=0.0553). According to in silico immunophenotyping, NLPHLs with splenic involvement were characterized by a lower proportion of naïve B cells and increased proportions of CD8+ T cells and M1-macrophages compared to the NLPHLs without splenic involvement (P<0.001 for all comparisons). Moreover, splenic involvement was associated with higher expression of checkpoint protein-encoding genes, including PDCD1 (PD-1), CD274 (PD-L1), CTLA4, LAG3, HAVCR2 (TIM-3) and TIGIT (Figure 1; P<0.001). Finally, we found a strong association between splenic involvement and gene expression profile related to inflammatory response (P<0.001), response to interferon gamma (P<0.001) and complement pathways (P<0.001). Conclusion: Our study represents the largest comprehensive clinical and transcriptomic analyses of NLPHL to date. The results demonstrate that NLPHLs with splenic involvement are characterized by inflamed TME with high expression of checkpoint molecule gene-signature and are associated with inferior outcomes. Figure 1. Unsupervised clustering for expression of checkpoint genes stratified by spleen involvement. Samples are ordered by the sum of normalized checkpoint gene expression values (checkpoint score) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

INTRODUCTION:We performed a single-center study of real-world health data to investigate the direct clinical consequence of targeted next-generation sequencing (NGS) results integrated in the clinicopathological evaluation of patients with cytopenia suspected of myelodysplastic syndrome (MDS).METHODS:The study included 87 newly referred patients, who had a bone marrow examination, which included targeted NGS analysis. NGS was requested at the discretion of either examining pathologist or hematologist. Data were collected retrospectively from patient files including pathology reports with integrated NGS results.RESULTS:The NGS results had a diagnostic impact in 67 cases (77%) when combining both histopathological and final clinical evaluation and provided prognostic value in 19 cases (22%). NGS supported a confident or tentative histopathological diagnosis in 52 cases (60%). Twenty cases (23%) had a final diagnosis of either Clonal Cytopenia of Undetermined Significance (CCUS) or Idiopathic Cytopenia of Undetermined Significance (ICUS). In 4 cases, NGS results affected the choice of principal treatment strategy, including considerations of allotransplantation. Twenty-one patients (24%) could be discharged to primary care physician.CONCLUSION:In a multidisciplinary clinicopathological real-world setting, NGS analysis of bone marrow samples from selected patients contributed substantially to the diagnostic evaluation and management of patients with cytopenia suspected of MDS. Consequently, we have now included NGS analysis in most routine bone marrow examinations from patients with MDS or unexplained cytopenia.

While the Danish Health Authority has withdrawn the vaxzevria vaccine from the Danish vaccination programme due to reports of rare cases of severe side effects, this case report presents a potential beneficial side effect of this vaccine. A patient presented, three days after her first jab, with localized itching and bleeding from a pigmented tumour on her lower leg, which was therefore excised, and, unexpectedly, diagnosed as a melanoma. We speculate if the vaccine-induced immune reaction resulted in increased immune activity within the tumour environment, and thus facilitated the early diagnosis of melanoma.

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

Bone involvement is a rare extranodal manifestation in patients with malignant lymphoproliferative diseases and has also been noted as a rare event in patients with Waldenstrom macroglobulinemia (WM). However, the actual prevalence has not been previously reported. We describe an unusual case of a patient with WM who presented with lower back pain and focal bone lesions at initial diagnosis. Magnetic resonance imaging (MRI) revealed multiple vertebral fractures. Positron emission tomography (PET) detected only nodal changes without pathological skeletal-related metabolic activity. Lymph node and bone marrow biopsies combined with an immunoglobulin M (IgM) M component revealed the diagnosis of WM. A next-generation sequencing (NGS) analysis using a targeted lymphoma panel of 59 recurrently mutated genes in lymphoid neoplasms showed mutations in the MYD88 and CD79B genes. After treatment with rituximab and bendamustine, the patient achieved a partial remission and pain relief. After 3 years of stable disease, a spontaneous subcapital fracture at the base of the femoral neck and new vertebral compression fractures occurred. Whole-body low-dose computed tomography (WB-LDCT) and bone density (dual energy X-ray absorptiometry (DEXA)) scan revealed marked osteopenia. After insertion of a hip prosthesis, examination of the removed hip showed infiltration of clonal lymphoplasmacytic cells. Our case confirms that one must be aware that bone involvement in patients with WM can occur as a rare manifestation. Interestingly, the MYD88/CD79B-mutated (MCD) genotype in diffuse large B-cell lymphoma is characterized by extranodal involvement and may also be involved in the pathogenesis of skeletal-related disease in the present case. As a follow-up to this unusual case, we have carried out an analysis based on the Danish Lymphoma Registry (LYFO) covering the entire national population in the period 2000 - 2020. The registry study included a cohort of 2,459 patients with WM and lymphoplasmacytic lymphoma. Our data revealed that primary bone involvement at diagnosis occurs in 1.75% of adults with WM. To the best of our knowledge, this is the first report of the prevalence of skeletal-related disease in a large nationwide cohort and defines bone involvement as an exceedingly rare event in WM.

Inactivating mutations in Bruton's tyrosine kinase (BTK) in patients with follicular lymphoma (FL) have recently been reported. These mutations were found in BTK inhibitor-treatment naïve patients. Here, we report the BTK mutation status in a real-world cohort of patients with non-Hodgkin lymphoma. We found primary BTK mutations in 7.7% of patients with large B-cell lymphoma (LBCL) and in 14.1% of patients with FL. All patients with BTK-mutated LBCL were BCL2 translocation positive, and the correlation between BCL2 translocation and BTK mutation persisted even when patients with known transformation from FL were excluded.

Objectives Postoperative endothelial damage potentially results in increased vascular leakage, tissue edema and subsequent complications. The preventive effect of glucocorticoids on endothelial damage after surgery is sparsely described, including the relation between endothelial damage and the postoperative inflammatory response. Thus, we aimed to assess the preventive effect of high-dose glucocorticoids on postoperative endothelial damage, and the association between endothelial damage and inflammation after surgery. Methods This was a predefined substudy of a randomized double-blinded clinical trial of methylprednisolone 10 mg/kg (high dose) vs. dexamethasone 8 mg (low dose) in patients undergoing liver resection at Rigshospitalet, Copenhagen. In total 25 patients undergoing major liver resection (11 in the high-dose group and 14 in the low-dose group) were included. The primary outcome was changed in five endothelial biomarkers and the secondary outcome was changes in inflammation [C-reactive protein (CRP)] for the first three postoperative days. Results No statistically significant difference was found for any endothelial biomarkers postoperatively between the two groups (P > 0.15, for all). High-dose glucocorticoids significantly reduced CRP on day 3 compared to low-dose glucocorticoids [median difference on a postoperative day 3, 59.6 g/L, (84.2; 27.1), P < 0.002]. No significant correlation between endothelial damage and CRP levels was seen. Conclusions No significant effect of high- vs. low-dose glucocorticoids on development in endothelial biomarkers after major liver resection was observed. High-dose glucocorticoids reduce the inflammatory response though without correlation to endothelial damage. Future studies should assess the clinical impact of increased endothelial biomarkers for clinical perioperative outcomes.

Background Commercial myeloid next-generation sequencing (NGS) panels may facilitate uniform generation of raw data between laboratories. However, different strategies for data filtering and variant annotation may contribute to differences in variant detection and reporting. Here, we present how custom data filtering or the use of Oncomine extended data filtering improve detection of clinically relevant mutations with the Oncomine Myeloid Research Assay. Methods The study included all patient samples ( n = 264) analyzed during the first-year, single-site, clinical use of the Ion Torrent Oncomine Myeloid Research Assay. In data analysis, the default analysis filter was supplemented with our own data filtering algorithm in order to detect additional clinically relevant mutations. In addition, we developed a sensitive supplementary test for the ASXL1 c.1934dupG p.Gly646fs mutation by fragment analysis. Results Using our custom filter chain, we found 96 different reportable variants that were not detected by the default filter chain. Twenty-six of these were classified as variants of strong or potential clinical significance (tier I/tier II variants), and the custom filtering discovered otherwise undetected tier I/tier II variants in 25 of 132 patients with clinically relevant mutations (19%). The remaining 70 variants not detected by the default filter chain were classified as variants of unknown significance. Among these were several unique variants with possible pathogenic potential judged by bioinformatic predictions. The recently launched Oncomine 5.14 extended filter algorithm detects most but not all of the tier I/tier II variants that were not detected by the default filter. The supplementary fragment analysis for the ASXL1 c.1934dupG p.Gly646fs confidently detected a variant allele frequency of down to 4.8% (SD 0.83%). The assay also detected the ASXL1 c.1900_1922del23 mutation. Conclusion Detection of clinically relevant variants with the Oncomine Myeloid Research NGS assay can be significantly improved by supplementing the default filter chain with custom data filtering or the recently launched Oncomine 5.14 extended filter algorithm. Our accessory fragment analysis facilitates easy testing for frequent ASXL1 mutations that are poorly or not covered by the NGS assay.

Affiliations Department of Pharmacology & Therapeutics, University of Florida, Gainesville, FL 32610 USA. Department of Chemistry, University of Florida, Gainesville, FL 32611 USA. Proteomics and Mass Spectrometry Facility, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610 USA. Department of Biology, Genetics Institute, University of Florida, Gainesville, FL 32610 USA Institute of Molecular Medicine, College of Medicine and Center for Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan. Department of Medicine, University of Florida, Gainesville, FL 32610 USA. UF-Health Cancer Center, University of Florida, Gainesville, FL 32610 USA. Department of Pathology, Copenhagen University Hospital Herlev, DK-2730 Herlev, Denmark.