Citations

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of undifferentiated myeloid cells that are expanded and activated in pathological conditions and have the ability to potently suppress T-cell function and thereby contribute to immunosuppression and tumor progression. While there have been studies showing a role for MDSC in a variety of hematological malignancies, no data is available indicating that MDSCs contribute to the tumor progression in Waldenstrom's Macroglobulinemia (WM), an indolent lymphoma characterized by bone marrow (BM) infiltration of lymphoplasmacytic (LPL) cells and increased secretion of monoclonal IgM. In previous work, we have found increased GM-CSF and reduced arginine and cysteine in the BM microenvironment in WM. We hypothesized that this was due to the presence and activity of MDSCs in WM. BM aspirates from WM patients (n=17) were therefore processed to isolate LPL (CD19+/CD138+) cells from the rest of the BM cells (CD19-/CD138-). Sorted (CD19-/CD138-) cells from BM of patients with WM were studied with flow cytometry. Using a sequential gating strategy (lack of lineage markers, low levels of HLADR, CD33+, CD11b+) we identified a population of MDSCs that were then subdivided using CD14 and CD15 expression into total-, monocytic-, or granulocytic- MDSCs (m-MDSC, g-MDSC). We also analyzed unsorted BM cells using cytometry by time-of-flight (CyTOF) in order to further identify and phenotypically characterize the BM MDSC population in a group of WM patients with smoldering (asymptomatic) disease, symptomatic disease, or in remission post-treatment. BM samples from normal subjects were used as a control. Flow cytometry data showed significant higher numbers of MDSC subsets expressing PD-L1 and Arginase1 in WM patients when compared to the normal samples. BM cells from WM patients (n=18) then were compared to controls (n=11), and the absolute number of the total MDSC (p=0.05), m-MDSC (p=0.002), g-MDSC (p=0.02) was increased in WM specimens. When MDSCs from WM or normal monocytes from healthy controls were co-cultured with activated T-cells, the proliferation of activated T-cells in the presence of MDSCs from WM patients was impaired compared to controls, confirming the suppressive role of MDSCs. We then performed high dimensional analysis of the total BM MDSC cells using t-SNEand identified phenotypically distinct MDSC cell populations in the BM that were differentially present when healthy controls were compared to patients with smoldering WM or those with WM needing treatment. Specifically, WM patients needing treatment had increased numbers of a distinct MDSC population that was highly positive for CD163, and CD138. Moreover, conventional markers denoting m-MDSC and g-MDSC, such as CD14 and CD15, were highly expressed in all populations and their pattern of expression did not specifically define the MDSC subtypes, indicating that high dimensional phenotyping further details the MDSC sub-compartments beyond the conventional categorization of MDSC using conventional cytometry. In summary, we find that MDSCs are increased in the BM of WM patients compared to controls. MDSCs expressing CD163 and CD138 increase when WM patients become symptomatic and require therapy. Furthermore, MDSCs in the BM of WM patients suppress T-cell function and likely contribute to progression of the disease. MDSCs in the BM therefore present a therapeutic target that should be explored in WM patients. Disclosures Ansell: Bristol Myers Squibb: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; AI Therapeutics: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials.

The outcomes of lymphoma patients treated with PD1 inhibitors (PD1i) vary significantly across different histologies with objective responses rates that are around 70% in classical Hodgkin lymphoma, but significantly lower, around 10%, in indolent non-Hodgkin lymphomas (iNHL). This may be due in part to the inability of PD1i to restore the function of exhausted T-cells (Tex) in the tumor microenvironment (TME). PD1 is expressed on various T-cell subsets, including effector cells and Tex. In this study we sought to identify markers of exhaustion beyond PD1 that define T-cell subsets with high reactivation potential in marginal zone lymphoma (MZL). To define different stages of exhaustion in MZL, we developed and validated a mass cytometry (CyTOF) panel which integrates T-cell markers of lineage, differentiation, activation, suppression, cytokine production and transcription factors associated with exhaustion. We analyzed 35 MZL spleens obtained at diagnosis and 6 reactive spleens. We identified a population of CD8 cells in MZL that express TIM3 without PD1, compose 1.5% (range 0.14-4.88%) of total CD8 cells and expand with activation through the TCR (6.2% of CD8, range 0.6-14.2%). Prolonged cytokine exposure has been shown to induce exhaustion in healthy T-cells, and so to determine whether PD1-TIM3+ cells are Tex, we treated healthy donor T-cells with activating beads and IL12 and analyzed them by flow cytometry. We showed that PD1-TIM3+ cells expand with repetitive activation and IL12 treatment suggesting that they are Tex. Since Tex have been shown to have a distinct transcription profile compared to functional effector T-cells, we analyzed the expression of several transcription factors (TFs) that are upregulated in Tex by CyTOF and compared them to other subsets of T-cells. PD1-TIM3+ cells expressed higher levels of Eomes, Tbet, TOX and Helios compared to activated effector cells, confirming that they are Tex, but lower levels levels of the same TFs compared to PD1+TIM3+ cells, consistent with a less severely exhausted phenotype. To characterize the function of PD1-TIM3+ cells, we manually gated on subsets of CD8+ non-naïve cells that were stained with the CyTOF panel and compared the different subsets. We showed that PD1-TIM3+ cells arise from effector cells, maintain their polyfunctionality by producing cytokines and chemokines and have a unique cytokine production profile, including perforin, granzyme B and proinflammatory cytokines, such as IL17A. Importantly, PD1-TIM3+ cells produced higher levels of most cytokines compared to more severely Tex expressing both PD1 and TIM3 (Fig 1). We then performed tSNE analysis where similar objects are modeled by nearby points. We determined that PD1-TIM3+ cells cluster together, suggesting that they are phenotypically homogeneous cells in terms of function and expression of activation markers and are significantly different from PD1+TIM3+ cells (Fig 2). We finally blocked PD1 or TIM3 and analyzed the effect of each antibody blockade on function. We showed that TIM3 blockade was able to restore the production of most cytokines to higher degrees than PD1 blockade. In addition, TIM3 blockade led to upregulation of CD28, which is critical for the rescue of Tex through costimulation, while PD1 blockade did not. We then analyzed the clinical outcomes of all 34 MZL patients. The frequency of PD1-TIM3+ cells was lower among observed patients who failed to achieve an event-free survival (EFS) of 12 months (EFS12) (0.8% vs 1.7%, p=0.02) and among treated patients that failed an EFS of 24 months (EFS24) (0.9 vs 1.7%, p=0.03), when compared to patients that reached these endpoints. Higher levels of PD1-TIM3+ cells were also associated with a trend towards improved overall survival (OS). Five-year OS was 88% vs. 69% in patients with PD1-TIM3+ levels >2% and <2% of total CD8 cells respectively (p=0.1) (Fig 3). Our study provides insights into the mechanisms of failure of PD1i in MZL and identifies a novel population of Tex with high reactivation potential which is clinically relevant and could serve as a target for future immunotherapies in iNHL. Disclosures Anagnostou: American Society of Hematology, Mayo Clinic/Iowa Lymphoma SPORE, Mayo Clinic Immune Monitoring Core, Mayo Clinic Hematology Small Grant: Research Funding. Novak:Celgene Coorperation: Research Funding. Ansell:Mayo Clinic Rochester: Employment; Trillium: Research Funding; Seattle Genetics: Research Funding; Regeneron: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Mayo Clinic Rochester: Employment; Affimed: Research Funding; LAM Therapeutics: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding; LAM Therapeutics: Research Funding; Mayo Clinic Rochester: Employment; Bristol-Myers Squibb: Research Funding; Mayo Clinic Rochester: Employment; Trillium: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Trillium: Research Funding; Mayo Clinic Rochester: Employment; Regeneron: Research Funding; Regeneron: Research Funding; Mayo Clinic Rochester: Employment; Seattle Genetics: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Affimed: Research Funding; Trillium: Research Funding; Mayo Clinic Rochester: Employment; Bristol-Myers Squibb: Research Funding; LAM Therapeutics: Research Funding; Trillium: Research Funding; Regeneron: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding; Mayo Clinic Rochester: Employment; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Research Funding; Regeneron: Research Funding; Mayo Clinic Rochester: Employment; Regeneron: Research Funding; Trillium: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Regeneron: Research Funding; Affimed: Research Funding; LAM Therapeutics: Research Funding; Affimed: Research Funding; LAM Therapeutics: Research Funding; Regeneron: Research Funding; Affimed: Research Funding; Trillium: Research Funding; Bristol-Myers Squibb: Research Funding; LAM Therapeutics: Research Funding; Affimed: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Trillium: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding.

Introduction: New Diffuse Large B-cell (DLBCL) treatments remain a clinical need despite the success of rituximab combined with CHOP chemotherapy (RCHOP), which results in durable responses in 60-70% of patients. Those refractory to, or who relapse following, first-line therapy have a very poor outcome, with only 20% surviving beyond 5 years. Rationally-targeted frontline strategies are needed, especially for those with high-risk disease. Significant advances have been made in the genomic classification of DLBCL, but none have impacted the design of phase III trials for untreated DLBCL patients. Building on new genetic profiling studies to personalize clinical treatment, an NCI initiative, could allow clinicians to add targeted therapies to the RCHOP backbone based on individual tumor signatures. The phase II ECOG-ACRIN1412 trial, which compared RCHOP combined with lenalidomide (R2CHOP) versus RCHOP showed significantly superior event free survival (EFS) and overall survival benefits for those treated with R2CHOP. Herein, we report the profile of a high-risk ABC/non-GCB subset of DLBCL driven by genomic alterations in inflammatory genes that are susceptible to front-line R2CHOP, but continue to experience poor outcome with RCHOP alone. Methods: We studied a total of 196 DLBCL patients. 47 were treated with R2CHOP from an investigator-initiated, open-label, single-arm phase II study (NCT00670358). 149 were newly diagnosed DLBCL cases treated with RCHOP, or R-immunochemotherapy (herein called RCHOP), and followed prospectively through the Molecular Epidemiology Resource (MER) of the University of Iowa/Mayo Clinic SPORE that served as a contemporary cohort. Patients from each treatment group were divided based on their event free survival at 24 months (EFS24). DNA alterations within these populations were identified through whole exome sequencing (WES). Variants were analyzed for their presence in EFS24 achievement or failure groups in both RCHOP and R2CHOP. Both tumor and germline sequencing was performed for 47/47 R2CHOP cases and 49/149 RCHOP cases. Gene expression data from the PanCan panel of 730 B-cell-related genes was analyzed to determine gene expression profiles characteristic of the high-risk/R2CHOP-profile on 59 available non-GCB DLBCL cases (45 RCHOP; 14 R2CHOP). A two-sided comparative marker analysis T statistic test was applied to assess what genes displayed differential expression based on achieving EFS24 in both R2CHOP and RCHOP populations. Positive values represented associations with achieving EFS24 and negative values were associated with cases of EFS24 failure. Results: Three genes were enriched in the RCHOP cases that failed EFS24 but achieved EFS24 with R2CHOP among non-GCB patients: PIM1, SPEN, and MYD88 (L265P), herein referred to as Responder Alterations. In R2CHOP cases, patients with a Responder Alteration had a better overall EFS (P = 0.051) compared to wild type patients. In contrast, RCHOP treated patients with a Responder Alteration in their tumor had a significantly worse overall EFS (P = 0.0004) compared to patients without a mutation. Together, PIM1, SPEN, or MYD88 (L265P) mutations were present in 38.0% (30/79) of all non-GCB cases. Collective R2CHOP and RCHOP EFS24 differential gene expression T values were significantly different for 18 previously-defined signatures. The R2CHOP cases that achieved EFS24 were enriched for genes involved in cell cycle, JAK-STAT, cytokine signaling, and NF-κB pathways based on these signatures and ontology analyses. Lastly, cases with WES and PanCan data were analyzed together to observe differential gene expression patterns between cases with and without signature mutations. These data suggest that R2CHOP disrupts tumors reliant on IRF4, NF-κB, and STAT transcription factors, leading to a loss of proliferative feedback systems. Conclusions: Our combined analysis of DNA and RNA across R2CHOP and RCHOP treatment cohorts identifies a high-risk non-GCB phenotype that encompasses approximately 38% of non-GCB patients, is capable of sustaining JAK-STAT and NF-κB signaling, and is sensitive to R2CHOP. Our study lays the groundwork for a precision therapy approach in DLBCL in which DNA or RNA profiles can be used to identify patients early in treatment who may not benefit from the current standard of care, RCHOP, and who would benefit from the addition of lenalidomide or other targeted agents. Disclosures Gandhi: Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Ansell:LAM Therapeutics: Research Funding; Seattle Genetics: Research Funding; LAM Therapeutics: Research Funding; Seattle Genetics: Research Funding; Regeneron: Research Funding; Bristol-Myers Squibb: Research Funding; Affimed: Research Funding; Trillium: Research Funding; Trillium: Research Funding; Regeneron: Research Funding; Bristol-Myers Squibb: Research Funding; LAM Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol-Myers Squibb: Research Funding; Trillium: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Regeneron: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding; Mayo Clinic Rochester: Employment; Regeneron: Research Funding; Seattle Genetics: Research Funding; Mayo Clinic Rochester: Employment; Seattle Genetics: Research Funding; LAM Therapeutics: Research Funding; LAM Therapeutics: Research Funding; LAM Therapeutics: Research Funding; Regeneron: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Affimed: Research Funding; Mayo Clinic Rochester: Employment; Mayo Clinic Rochester: Employment; Mayo Clinic Rochester: Employment; Seattle Genetics: Research Funding; Mayo Clinic Rochester: Employment; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Regeneron: Research Funding; Bristol-Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Mayo Clinic Rochester: Employment; Mayo Clinic Rochester: Employment; Trillium: Research Funding; Affimed: Research Funding; Mayo Clinic Rochester: Employment; Bristol-Myers Squibb: Research Funding; Affimed: Research Funding; LAM Therapeutics: Research Funding. Cerhan:Celgene: Research Funding; NanoString: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Nowakowski:Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Genentech, Inc.: Research Funding; MorphoSys: Consultancy, Research Funding; NanoString: Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees. Novak:Celgene Coorperation: Research Funding.

Background: NGS of myeloid mutations is an integral part of AML clinical decision-making. There is currently no information regarding concordance between NGS panels in AML using samples from the same patient across various platforms in different diagnostic laboratories. To study this important question, we analyzed NGS of myeloid mutations in diagnostic samples from The Beat AML Master Trial (BAMT) for newly diagnosed older AML patients, and compared variant calls made between institutional laboratories enrolling the study subject with those made by Foundation Medicine (FM), the central laboratory used for treatment assignment in this precision medicine trial. Methods: We identified newly diagnosed AML patient samples (peripheral blood (PB) and/or bone marrow (BM)) from 2 lead institutions in the BAMT(Ohio State, OSU and Oregon Health and Sciences University, OHSU) that were analyzed by both the institutional and by FM from Nov 15, 2016 to Apr 15, 2019. Samples sent to both laboratories >3 days apart were excluded. Samples were analyzed at the institutional laboratories using their respective NGS mutational panels and by FM using the FoundationOne®Heme(FMH) NGS panel which utilizes capture based sequencing. The OSU NGS assay utilizes sequencing on Illumina MiSeq. The OHSU NGS assay employs semiconductor-based sequencing (Ion Torrent PGM platform). The variant allele frequency (VAF) sensitivity for detection for all 3 laboratories range from 1-2%. We evaluated the ability to identify mutations in 8 genes : FLT3, IDH1/ 2, NPM1, TET2, DNMT3A, WT1 and TP53 used in treatment assignment in theBAMT. A detection cutoff of 2% was used to define the presence or absence of a mutation. Overall, agreement was defined as the number of times the local and central laboratories made the same call divided by the total number of patients. Sensitivity was defined as the number of present calls made locally divided by the number of present calls made centrally, and specificity as the number of absent calls made locally divided by the number of absent calls made centrally. The overall kappa statistic, controlling for institution, provided another measure of agreement between local and central calls, where a value of 1 indicates perfect agreement. Results: 194 patient samples were identified using methods above and analyzed locally at the screening institution (125 at OSU, 69 at OHSU) and centrally at FM. Type of tissue analyzed for variants between local site and FM were 59 PB, 129 BM, and 6 with BM/PB mismatch. Overall agreement in presence/absence calls between local and central results for each of the 8 genes was over 95% (Table 1). There was perfect agreement for NPM1. The sensitivity was above 94% for all genes except TP53 (88.6%) and WT1 (63.6%). Failure to detect a mutation locally was primarily due to reporting of all TP53 variants, including variants of unknown significance (VUS) (5) by FM as agreed upon in the study protocol, detection at low levels below local site sensitivity cutoff (1), detection of variants in a portion of gene not covered at the local site(1)and possible artifact (1). For the WT1 gene, discordance in 5 samples included VUS (3) reported by FM ,a variant detected in a portion of the gene not covered at the local site(1).and difference in leukemic tissue analyzed with mutation not detected by the central laboratory on a PB sample, and present at the institutional lab on a BM sample; affecting the overall agreement and specificity but not sensitivity. Specificity was at least 98% for each of the 8 genes. Finally, most discrepancies in reported mutations in FLT3 (n=2), IDH1 (n=1), IDH2 (n=2), DNMT3A (n=4) and TET2 (n=5) were due to reporting of VUS in one laboratory and not by another. Conclusion: Detection of pathogenic myeloid mutations using orthogonal assays showed a high degree of concordance for genes used in therapeutic assignment on the BAMT.The small number of discordant results, in TP53 and WT1, were attributed to the reporting of VUS. This study illustrates the importance of quality control and standardization as NGS continues to be widely utilized in AML for clinical decision making, with a variety of platforms across multiple laboratories. Our next steps involve evaluating the differences in VAFs reported between local and central laboratories when a given mutation is identified, as well as the potential reasons for observed differences and clinical implications of known pathogenic mutations vs putative VUS. Disclosures Borate: Daiichi Sankyo: Consultancy; AbbVie: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Pfizer: Consultancy. Vergilio:Foundation Medicine: Employment; Roche Holding AG: Equity Ownership. Stein:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Patel:France Foundation: Honoraria; Dava Oncology: Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Astellas: Research Funding; Abbvie: Research Funding; AI Therapeutics: Research Funding; Forma: Research Funding; Incyte: Research Funding; Kite: Research Funding; Takeda: Research Funding. Stock:Daiichi: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; UpToDate: Honoraria; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Schiller:Amgen: Other, Research Funding; Astellas: Research Funding; Biomed Valley Discoveries: Research Funding; Bristol Myer Squibb: Research Funding; Celgene: Research Funding, Speakers Bureau; Constellation Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding; Eli Lilly and Company: Research Funding; FujiFilm: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; Incyte: Research Funding; J&J: Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Karyopharm: Research Funding; Novartis: Research Funding; Onconova: Research Funding; Pfizer Pharmaceuticals: Equity Ownership, Research Funding; Sangamo Therapeutics: Research Funding; Agios: Research Funding, Speakers Bureau. Blum:AmerisourceBergen: Consultancy; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Astellas,: Research Funding; Xencor: Research Funding; Forma: Research Funding. Kovacsovics:Pfizer: Research Funding; Jazz: Consultancy; Novartis: Research Funding; Abbvie: Research Funding; Amgen: Consultancy, Research Funding. Foran:Agios: Honoraria, Research Funding. Druker:Pfizer: Research Funding; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Cepheid: Consultancy, Honoraria; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding. Byrd:Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S. Levine:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Prelude Therapeutics: Research Funding; Novartis: Consultancy; Gilead: Consultancy; Lilly: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees. Mims:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Abstract We report a case of an adult male patient with multifocal urinary bladder paragangliomas, which were negative on 68Ga-DOTATATE PET/CT scan, but positive on 123I-MIBG SPECT/CT scan. While the 68Ga-DOTA analog PET/CT exhibits superior performance in diagnosis and staging of pheochromocytoma and paraganglioma, our case demonstrates negative somatostatin receptor expression in this rare entity and indicates that 123I-MIBG SPECT/CT still plays a vital role in characterization of bladder paraganglioma.

Rapid advances in technology are revealing previously unknown organization, cooperation, and limitations within the population of nontumor cells surrounding the tumor epithelium known as the tumor microenvironment (TME). Nowhere are these findings more pertinent than in the gastrointestinal (GI) tract where exquisite cell specialization supports a complex microenvironmental niche characterized by rapid stemness-associated cell turnover, pathogen sensing, epithelial orchestration of immune signaling, and other facets that maintain the complex balance between homeostasis, inflammation, and disease. Here, we summarize and discuss select emerging concepts in the precancerous microenvironment, TME, and tumor epithelial-TME crosstalk as well as their implications for the management of GI cancers.

OBJECTIVES: Preoperative decision-making for differentiating malignant from benign lesions in the gallbladder remains challenging. We aimed to create a diagnostic nomogram to identify gallbladder cancer (GBC), especially for incidental GBC (IGBC), before surgical resection. METHODS: A total of 587 consecutive patients with pathologically confirmed gallbladder lesions from a hospital were randomly assigned to a training cohort (70%) and an internal validation cohort (30%), with 287 patients from other centers as an external validation cohort. Radiological features were developed by the least absolute shrinkage and selection operator logistic regression model. Significant radiological features and independent clinical factors, identified by multivariate analyses, were used to construct a nomogram. RESULTS: A diagnostic nomogram was established by age, CA19.9, and 6 radiological features. The values of area under the curve in the internal and external validation cohorts were up to 0.91 and 0.89, respectively. The calibration curves for probability of GBC showed optimal agreement between nomogram prediction and actual observation. Compared with previous methods, it demonstrated superior sensitivity (91.5%) and accuracy (85.1%) in the diagnosis of GBC. The accuracy using the nomogram was significantly higher in GBC groups compared with that by radiologists in the training cohort ( P < 0.001) and similarly in each cohort. Notably, most of the IGBC, which were misdiagnosed as benign lesions, were successfully identified using this nomogram. DISCUSSION: A novel nomogram provides a powerful tool for detecting the presence of cancer in gallbladder masses, with an increase in accuracy and sensitivity. It demonstrates an unprecedented potential for IGBC identification.

Abstract Background Polymerase chain reaction (PCR)/sequencing targeting the 16S ribosomal RNA (rRNA) gene to detect bacteria in normally sterile tissues and fluids has become increasingly popular in clinical medicine. This culture-independent technique can detect bacteria that are nonviable or difficult to cultivate using conventional methods. The clinical value of this type of testing is not well defined. We aimed to assess the diagnostic value of 16S rRNA PCR/Sanger sequencing as a clinical diagnostic assay at Mayo Clinic. Methods This is an interim analysis of the first 173 of 478 patients who had 16S rRNA PCR/Sanger sequencing done on sterile tissues or fluids at our institution from April, 2017 to November, 2018 as part of routine clinical practice. Medical records are being retrospectively reviewed, with results compared with those of culture. Results We reviewed 207 specimens from 173 patients (musculoskeletal 79%, cardiovascular 7%, central nervous system 4%, other 9%) that underwent 16S rRNA PCR/Sanger sequencing by clinical request (Table 1). In 90% of these specimens, the test was pre-planned rather than added-on. Nine specimens were excluded from analysis, as cultures were not performed. Overall concordance of culture with PCR/sequencing was 81% (160/197; P < 0.0001). Of 44 culture-positive specimens, PCR detected the same bacterium in 21 (48%) (Table 2). 45% (20/44) of those with positive cultures and 46% of those with positive PCR/sequencing results had received prior antimicrobial therapy (Table 3). PCR was negative in 139/144 specimens that were culture-negative (97%). PCR/sequencing was helpful in detecting a putative bacterial pathogen in 4 patients with negative cultures (Table 4). Conclusion Overall, 16S rRNA PCR/Sanger sequencing improved diagnostic yield compared with culture in a minority of cases. The described assay is limited by its inability to detect polymicrobial infections, a technical limitation that could possibly be addressed using massive parallel sequencing. Careful selection of cases and a save and add-on approach may be more cost-effective than upfront testing, although this was requested in a minority of cases. Disclosures Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Growing evidence supports gene fusions as good candidates for molecularly targeted therapy in CRC. Here we describe a case of a 63-year-old man who had a radical right hemicolectomy procedure 24 months ago. Pathological diagnosis indicated colorectal adenocarcinoma with stage pT4N2bMx. During re-examination in December 2016, positron emission tomography/computed tomography scans indicated relapse with multiple lymph nodes metastasis. Then the patient received a nine-cycle combination treatment of XELOX and bevacizumab and showed progressive disease (PD). Subsequently, the patient was treated with bevacizumab plus FOLFIRI for 2 months before discontinuation because of adverse events. Paraffin sections of postoperative colorectal tissue were subjected to next-generation sequencing, and epidermal growth factor receptor (EGFR) amplification and rare EGFR–SEPT14 fusion were identified. The patient then received erlotinib, an EGFR tyrosine kinase inhibitor (TKI), and achieved a partial response. However, the patient subsequently showed PD, and a new variant, EGFRvIII, appeared in metastasis, which may be involved in erlotinib resistance. We suggest that there is value in treating patients harboring EGFR fusions with EGFR TKI therapy, and EGFR–SEPT14 fusion may be used as a therapeutic target for CRC. Key Points To the authors' knowledge, this is the first report of EGFR–SEPT14 fusion in colorectal cancer. The patient achieved a partial response after treatment with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. This report expands the list of gene fusions in colorectal cancer and highlights new targets for the therapeutic intervention. EGFRvIII may be involved in erlotinib resistance, which is rare in colorectal cancer.