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Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by inter-species differences between human and mouse, as well as inter-person germline and somatic variation. Here we develop an autologous system that models the TME in individual patients. With patient-derived bone marrow, we engrafted a patient’s hematopoietic system in MISTRG6 mice followed by patient-derived xenograft (PDX) tissue, providing a genetically matched autologous model. We used this system to prospectively study tumor-immune interactions in solid tumor patients. Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent VEGF-A signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth, demonstrating the utility of the autologous PDX system for pre-clinical testing.

The immune milieu within tumors, consisting of diverse cell types including adaptive immune cells as well as macrophages, dendritic cells, natural killer and other innate immune cells, is critical to determining cancer outcome. However, the immune tumor microenvironment (TME) has been challenging to model, owing to inherent inter-species differences. While humanized mice can support human immune cells, the hematopoietic stem and progenitor cells (HSPCs) used for transplantation have been largely limited to fetal or neonatal stem cell sources, necessitating allogeneic experiments with limited applicability. We sought to develop a method to pre-clinically model an individual adult cancer patient, capturing the unique features of an individual such as germline genetic determinants of immune function and somatic tumor heterogeneity, and creating an autologous system. MISTRG6 may be engrafted with low numbers of HSPCs. When engrafted with equivalent numbers of CD34+ cells from human fetal liver (FL), neonatal cord blood (CB), adult mobilized peripheral blood (MPB), or adult bone marrow (BM), MISTRG6 mice harbored greatly increased human hematopoietic cells as a proportion of total hematopoietic cells in peripheral blood compared with NOD-scid-gamma (NSG) and MISTRG mice (p MISTRG6 allows efficient engraftment of patient derived HSPCs. We sought to apply this improved engraftment prospectively to model individual patients’ TME through collection of BM-derived CD34+ cells from patients under active treatment along with tumor tissue from the same patient. At two cancer centers, we enrolled patients with melanoma, NSCLC, PDAC, and HNSCC to provide BM aspirate, peripheral blood, and tumor tissue. CD34+ cells were isolated from BM aspirates and tumor tissue was utilized to generate PDXs. Overall, 71 patients were enrolled, 46 melanoma, 19 NSCLC, 4 PDAC, 2 HNSCC, ages 22-85, 39% females. These yielded autologous, immune-reconstituted MISTRG6 hosts from 14 melanoma, 5 NSCLC, 2 PDAC, and 1 HNSCC patients. Autologously engrafted MISTRG6 mice displayed the gamut of human immune cells of adaptive and innate types in PB at 7 weeks of age. Notably, this included CD33+ myeloid cells such as CD14+CD16− classical, CD14+CD16+ intermediate, and CD14−CD16+ non-classical monocytes. Moreover, human dendritic cells (DCs), key innate immune cells for initiation of anti-tumor responses were readily detected by flow cytometry in spleens of autologously-engrafted mice, including cDC1, cDC2, and pDC cells. MISTRG6 mice bearing a patient’s hematopoietic cells support autologous PDX growth. Having achieved successful engraftment of patient hematopoietic systems in MISTRG6 hosts, we next subcutaneously introduced the patient’s matched PDX tumor tissue to generate autologously engrafted PDX mice. For most patients, tumors grown in autologous HSPC-engrafted hosts were significantly larger than in non-engrafted hosts. Multicolor immunofluorescence staining of PDX tumors demonstrated that human immune cells, including CD3+ T cells, CD14+ and HLA-DR+ myeloid cells, penetrated deeply into the tumor and co-localized with tumor cells as well as with other engrafted immune cells. Indeed, HLA-DR+CD14+macrophages and HLA-DR+CD14(-) dendritic cells were present, and direct physical interaction between T cells and macrophages was evident. Using whole-exome sequencing, we found that 225 somatic changes were shared between patient Mel738’s surgical resection sample, two PDX tumors from non-engrafted mice lacking human immune cells, and two PDX tumors from mice with autologous engraftment. 5 additional changes were shared among the tumor samples and absent from the cell line, with 36 additional mutations being specific to the cell line. These data underscore the capacity of the autologous PDX method to recapitulate the somatic heterogeneity that the patient tumor possesses. Autologous MISTRG6 mice display diverse human immune cell populations and recapitulate an immunosuppressive TME. To fully characterize the autologous MISTRG6 model and investigate mechanisms by which autologous human immune cells enhance tumor growth, we performed single cell transcriptomics on hCD45+-enriched cells from blood and tumor isolated from autologous mice. This revealed 16 distinct cell subtypes, including 3 myeloid, 2 NK cell, 2 CD8 T cell, 3 CD4 T cell, 2 cycling lymphocyte, 1 B cell, and 3 melanoma cell clusters. Subclustering of myeloid cells revealed 9 distinct clusters including 4 monocyte, 4 macrophage, and 1 DC cluster. Comparing CD8 T cells present in blood versus tumor revealed that the most differentially expressed genes (DEGs) found in blood were characteristic of naïve T cells, while genes present in the TME were consistent with activated T cell phenotypes. In addition, sub-clustering revealed 3 distinct CD8 T cell types that included two activated-like populations, with one of these populations also expressing an activated/exhausted program typified by expression of PDCD1, LAG3, and GZMA. Naïve-like T cells were most highly represented in the blood, while activated and activated/exhausted-like genes were more present in the TME. Inhibiting the actions of human VEGF-A blocks the enhanced tumor growth in autologously engrafted mice. Notably, IPA Upstream Regulator Analysis identified VEGFA, a central player in tumor growth and vascularization, as a key upstream inducer of genes in the TME (FDR p= 5.65 × 10−13). Indeed, expression VEGFA itself was nearly absent in blood but induced in the TME, especially in macrophages and VEGFA targets were highly represented among the DEGs between tumor and blood.To test the relevance of VEGF-A in the TME, we selectively blocked human VEGF-A by treating autologous mice humanized from Mel2 with the anti-hVEGF-A antibody bevacizumab that has high affinity for human VEGF-A yet low affinity for mouse VEGF-A. PDXs grown in untreated autologously engrafted MISTRG6 mice grew significantly larger than those in non-engrafted littermate control hosts (p Future Directions: Thus, these in silico and in vivo results suggest that human VEGF-A production in the autologous TME enhances tumor growth in MISTRG6 PDX models and underscores the utility of the MISTRG6 system for pre-clinical testing of drugs that act on human immune components of the TME. By engrafting mice with bone marrow derived stem cells followed by implantation of tumor derived from the same donor, we have demonstrated that autologous MISTRG6 models recapitulate important features of the human TME, including sufficient immunosuppression to prevent tumor clearance, presence of activated/exhausted T cells, and innate immune cells including DCs, monocytes, NK cells, and macrophages, the latter especially relevant to the production of VEGF-A. Citation Format: Michael Chiorazzi, Jan Martinek, Bradley Krasnick, Yunjiang Zheng, Keenan Robbins, Rihao Qu, Gabriel Kaufmann, Zachary Skidmore, Laura Henze, Frederic Brösecke, Adam Adonyi, Jun Zhao, Liang Shan, Esen Sefik, Jacqueline Mudd, Ye Bi, S Peter Goedegebuure, Malachi Griffith, Obi Griffith, Abimbola Oyedeji, Sofia Fertuzinhos, Roland Garcia-Milian, Daniel Boffa, Frank Detterbeck, Andrew Dhanasopon, Justin Blasberg, Benjamin Judson, Scott Gettinger, Katerina Politi, Yuval Kluger, A Karolina Palucka, Ryan Fields, Richard A. Flavell. Autologous humanized PDX modeling for immuno-oncology recapitulates the human tumor microenvironment. [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 NG11.

Current understanding of tumor immunosuppressive mechanisms forms the basis for modern day immunotherapies. Immunoregulatory role of platelets in cancer remains largely elusive. Platelets from non-small cell lung cancer (NSCLC) patients revealed a distinct activation phenotype. TREM-like transcript 1 (TLT-1), a platelet protein, was increased along with enhanced extracellular release from NSCLC platelets. The increased platelet TLT-1 was also evident in humanized mice with patient-derived tumors. In immunocompetent mice with syngeneic tumors, TLT-1 binding to T cells, in vivo, led to suppression of CD8 T cells, promoting tumor growth. We identified direct interaction between TLT-1 and CD3ε on T cells, implicating the NF-κB pathway in CD8 T cell suppression. Anti–TLT-1 antibody rescued patients’ T cells from platelet-induced suppression ex vivo and reduced tumors in mice in vivo. Clinically, higher TLT-1 correlated with reduced survival of NSCLC patients. Our findings thus identify TLT-1 as a platelet-derived immunosuppressor that suppresses CD8 T cells and demonstrate its therapeutic and prognostic significance in cancer.

While several clinical and immunological parameters correlate with disease severity and mortality in SARS-CoV-2 infection, work remains in identifying unifying correlates of coronavirus disease 2019 (COVID-19) that can be used to guide clinical practice. Here, we examine saliva and nasopharyngeal (NP) viral load over time and correlate them with patient demographics, and cellular and immune profiling. We found that saliva viral load was significantly higher in those with COVID-19 risk factors; that it correlated with increasing levels of disease severity and showed a superior ability over nasopharyngeal viral load as a predictor of mortality over time (AUC=0.90). A comprehensive analysis of immune factors and cell subsets revealed strong predictors of high and low saliva viral load, which were associated with increased disease severity or better overall outcomes, respectively. Saliva viral load was positively associated with many known COVID-19 inflammatory markers such as IL-6, IL-18, IL-10, and CXCL10, as well as type 1 immune response cytokines. Higher saliva viral loads strongly correlated with the progressive depletion of platelets, lymphocytes, and effector T cell subsets including circulating follicular CD4 T cells (cTfh). Anti-spike (S) and anti-receptor binding domain (RBD) IgG levels were negatively correlated with saliva viral load showing a strong temporal association that could help distinguish severity and mortality in COVID-19. Finally, patients with fatal COVID-19 exhibited higher viral loads, which correlated with the depletion of cTfh cells, and lower production of anti-RBD and anti-S IgG levels. Together these results demonstrated that viral load – as measured by saliva but not nasopharyngeal — is a dynamic unifying correlate of disease presentation, severity, and mortality over time.

Here we analyzed patient samples of metastatic melanoma, and found that tumor-infiltrating T cells are primarily juxtaposed to CD14+ monocytes/macrophages rather than melanoma cells. Using novel immunofluorescence-guided laser capture microdissection, we analyzed transcriptomes of CD3+ T cells, CD14+ monocytes/macrophages and melanoma cells in non-dissociated tissue. CD14+ cells localized within tumor nests displayed a specific transcriptional signature distinct from CD14+ cells localized in tumor stroma. When applied to TCGA cohorts, this stromal macrophage gene set distinguished patients with significantly prolonged survival in cutaneous melanoma and as well as other cancers. This signature contains CD2 and Ly75, a gene linked with antigen capture and regulation of tolerance and immunity, and comprises markers of monocyte-derived dendritic cells (DCs). Thus, the stromal CD14+ cell signature represents a novel candidate biomarker and suggests that reprogramming of stromal macrophages to acquire DC function may offer a therapeutic opportunity for numerous metastatic cancers.

Modulation of immune function at the tumor site could improve patient outcomes. Here, we analyze patient samples of metastatic melanoma, a tumor responsive to T cell-based therapies, and find that tumor-infiltrating T cells are primarily juxtaposed to CD14

Rationale and Objective Acute kidney injury (AKI) is common in patients with COVID-19 and associated with poor outcomes. Urinary biomarkers have been associated with adverse kidney outcomes in other settings and may provide additional prognostic information in patients with COVID-19. We investigated the association between urinary biomarkers with adverse kidney outcomes among patients hospitalized with COVID-19. Study Design Prospective cohort study. Setting and Participants Patients hospitalized with COVID-19 (n=153) at 2 academic medical centers between April and June 2020. Exposures 19 urinary biomarkers of injury, inflammation, and repair. Outcomes Composite of KDIGO stage 3 AKI, requirement for dialysis, or death within 60 days of hospital admission. We also compared various kidney biomarker levels in the setting of COVID-19 versus other common AKI settings. Analytic Approach Time-varying Cox proportional hazards regression to associate biomarker level with composite outcome. Results Out of 153 patients, 24 (15.7%) experienced the primary outcome. Two-fold higher levels of neutrophil gelatinase-associated lipocalin (NGAL) (HR: 1.34; 95% CI: 1.14-1.57), monocyte chemoattractant protein (MCP-1) (HR: 1.42; 95% CI: 1.09-1.84), and kidney injury molecule-1 (KIM-1) (HR: 2.03; 95% CI: 1.38-2.99) were associated with highest risk of sustaining primary composite outcome. Higher epidermal growth factor (EGF) levels were associated with a lower risk of the primary outcome (HR 0.61; 95% CI: 0.47-0.79). Individual biomarkers provided moderate discrimination and biomarker combinations improved discrimination for the primary outcome. The degree of kidney injury by biomarker level in COVID-19 was comparable to other settings of clinical AKI. There was evidence of subclinical AKI in COVID-19 patients based on elevated injury biomarker level in patients without clinical AKI defined by serum creatinine. Limitations Small sample size with low number of composite outcome events. Conclusion Urinary biomarkers are associated with adverse kidney outcomes in patients hospitalized with COVID-19 and may provide valuable information to monitor kidney disease progression and recovery., Graphical abstract

This article describes what we believe to be the first course to teach future law librarians how to best teach legal research. It was a process of trial and error and has evolved over the years. Th...

Cancer patients display immunomodulation related to malignancy and anti-cancer therapies, but how these factors impact COVID-19 remains unknown. To investigate immune responses in cancer patients with COVID-19, we undertook a prospective case-control study, enrolling hospitalized solid tumor patients with acute COVID-19, as well as age-, gender-, and comorbidity-matched COVID-19 patients without cancer as controls. Using biospecimens collected during hospitalization, we performed virologic measurements as well as in-depth immunophenotyping of cellular, antibody and cytokine responses. We enrolled 17 cancer patients (cases) admitted to Yale-New Haven Hospital between March 15 and June 30, 2020 with COVID-19, as well as 17 matched non-cancer patients (controls) admitted with COVID-19. No significant differences were observed between cases and controls based on patient characteristics (age, gender, race, co-morbidities, smoking history, days from symptom onset to COVID-19 diagnosis) or outcomes (COVID-19 severity, length of hospital stay, rate of intubation or mortality). The most common primary tumor sites were lung (4/17) and gastrointestinal (4/17); all cases had received cancer-directed therapy within 6 months of COVID-19 diagnosis, with 13/17 receiving treatment less than 1 month prior to hospitalization. Three of 17 cases had received immune checkpoint inhibitor therapies. Despite having similar SARS-CoV-2 viral RNA loads at the time of COVID-19 diagnosis when compared with controls, cancer cases had increased viral RNA abundance during hospitalization, suggesting slower clearance. Antibody responses against SARS-CoV-2 were preserved in cancer cases, with cases displaying similar levels of IgM and IgG antibodies directed against SARS-CoV-2 epitopes compared to controls. Cytokine profiling revealed higher plasma levels of CCL3, IL1A and CXCL12 in cancer cases compared to controls. Using flow cytometric immunophenotyping, we found that innate immune and non-T cell adaptive immune parameters were similar between cases and controls hospitalized with COVID-19. However, among cancer cases on conventional therapies, T cell lymphopenia was more profound, and these cases demonstrated higher levels of CD8+ exhausted (CD8+CD45RA−PD1+TIM3+), CD8+GranzymeB+ and CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ activated T cells when compared with controls; interestingly, these differences were not observed in patients who had received immune checkpoint inhibition. Thus, we found reduced viral RNA clearance and specific alterations in T cell and cytokine responses in cancer patients hospitalized with COVID-19 compared with matched controls with COVID-19. This dysregulated T cell response in cancer patients, which may reflect immune modulation due to chronic antigen stimulation as well as cancer therapies, may lead to altered virologic and clinical outcomes in this population. Citation Format: Michael Chiorazzi, Erin Silva, Kristina Brower, Patrick Wong, Carolina Lucas, Jon Klein, Feimei Liu, Maura Nakahata, Jun Zhao, Nur-Taz Rahman, Camila Odio, Santos Bermejo, Shelli F. Farhadian, Charles Dela Cruz, Arnau Casanovas-Massana, John Fournier, Catherine Muenker, Anne L. Wyllie, Chantal B.F. Vogels, Chaney C. Kalinich, Mary E. Petrone, Isabel M. Ott, Anne E. Watkins, Adam J. Moore, Tara Alpert, Yuval Kluger, Aaron Ring, Nathan D. Grubaugh, Akiko Iwasaki, Albert I. Ko, Roy S. Herbst. Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr S03-03.

Mechanisms contributing to immunotherapy resistance in patients are the object of intense studies. The myeloid cells play a major role in tumors and include cells with different functions that can be grossly summarized as: (1) Antigen capture for presentation (dendritic cells, DCs) or for degradation (macrophages); (2) Tissue repair (macrophages) and (3) effector function (mast cells, monocytes, granulocytes). However, the functional status of myeloid cells in human tumors is not completely understood. To study intact tumor microenvironments, we have established a comprehensive approach for cellular and molecular analysis. Polychromatic immunofluorescence and histocytometry showed that CD14+ cells represent the majority of the total tumor immune infiltrate. Furthermore, while majority of the T cells are located outside of cancer nests, most of the T cells present in the melanoma tissue are in direct contact with CD14+ cells rather than melanoma cells. The distribution of CD14+ cells shows two distinct patterns: CD14+ cells within cancer nest (intratumoral) are in close interactions with melanoma cells and are loaded with melanoma protein; CD14+ cells in the tissue surrounding cancer nest (stromal) do not show melanoma protein cargo. Using customized immunofluorescence guided laser capture micro-dissection, we harvested CD3+ T cells based on their tissue location and CD14+ cells based on their tissue location and melanoma protein load for downstream analysis. Both stromal and intratumoral CD14+ cells display a macrophage like identity with RNA expression of so called “M2 like” macrophages markers, yet we detect a significant degree of variability in the expression levels for certain markers. Transcriptional profiling showed that CD14+ cells clustered according to their tissue localization, i.e., intratumoral vs. stromal, interestingly this localization imprint was less pronounced for T cells, where samples clustered preferentially by patient, leading to much lower number of localization specific differentially expressed genes. Computational analysis, revealed distinct gene signatures associated with different inflammatory and metabolic pathways in intratumoral and stromal CD14+ cells. Thus, transcriptome differentiates functional status of CD14+ cells related to their localization within tumor. Further the intra/stromal CD14+ signature clusters patient with significantly better long term survival across multiple cancer types in TCGA, which in metastatic melanoma was linked with dendritic cells signature in the stroma. Finally, combining our CD3+ and CD14+ LCM data we identified localization specific pairs of receptor/ligand interactions between myeloid and T cells. Citation Format: Jan Martinek, Kyung In Kim, Jianan Lin, Te-Chia Wu, Hannah Borouchov, Ananya Gulati, Lili Sun, Victor Wang, Joshy George, Philipp Henrich, Florentina Marches, Anthony Rongvaux, Michael Chiorazzi, Jeffrey H. Chuang, Paul Robson, Richard Flavell, Jacques Banchereau, Karolina Palucka. Transcriptional profiles of CD14+ cells in situ in melanoma reveal plasticity, localization dependent function and specific T cell interactions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3441.

Mechanisms contributing to immunotherapy resistance in patients are the object of intense studies. The myeloid cells play a major role in tumors and include cells with different functions that can be grossly summarized as (1) antigen capture for presentation (dendritic cells, DCs) or for degradation (macrophages), (2) tissue repair (macrophages), and (3) effector function (mast cells, monocytes, granulocytes). However, the functional status of myeloid cells in human tumors is not completely understood. To study intact tumor microenvironments, we have established a comprehensive approach for cellular and molecular analysis. Polychromatic immunofluorescence and histocytometry showed that CD14+ cells represent the majority of the total tumor immune infiltrate. Furthermore, while the majority of the T cells are located outside of cancer nests, most of the T cells present in the melanoma tissue are in direct contact with CD14+ cells rather than melanoma cells. The distribution of CD14+ cells shows two distinct patterns: CD14+ cells within cancer nest (intratumoral) are in close interactions with melanoma cells and are loaded with melanoma protein; CD14+ cells in the tissue surrounding cancer nest (stromal) do not show melanoma protein cargo and are organized in dense infiltrates. Using customized immunofluorescence-guided laser capture microdissection, we harvested CD3+ T cells based on their tissue location and CD14+ cells based on their tissue location and melanoma protein load for downstream analysis. RNAseq revealed transcriptional profiles that clustered patient samples according to their tissue localization, i.e., intratumoral vs. stromal; interestingly, this was not the case for the CD3+ T cells. Computational analysis revealed distinct gene signatures associated with different pathways in intratumoral and stromal CD14+ cells. Thus, transcriptome differentiates functional status of CD14+ cells related to their localization within tumor. Further, the intra/stromal CD14+ signature clustered TCGA patient samples with significantly different survival, which in metastatic melanoma was linked with dendritic cells’ signature in the stroma. Finally, we show that receptor ligand interactions between myeloid and T cells are tissue localization specific. Citation Format: Jan Martinek, Kyung in Kim, Te-Chia Wu, Hannah Boruchov, Ananya Gulati, Lili Sun, Victor Wang, Joshy George, Philipp Henrich, Florentina Marches, Anthony Rongvaux, Michael Chiorazzi, Jeff Chuang, Paul Robson, Richard Flavell, Jacques Banchereau, Karolina Palucka. Transcriptional profiles of CD14+ cells in situ in melanoma reveal plasticity, localization-dependent function, and specific T-cell interactions [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A103.

The tumor microenvironment presents metabolic constraints to immunosurveiling cells. In this issue of Cancer Cell, Zhang et. al. demonstrate that CD8(+) TILs reprogram under hypoxic and hypoglycemic conditions, regaining effector function by engaging fatty acid catabolism, which is promoted by fenofibrate and synergistic with immune checkpoint blockade therapy.

Mechanisms contributing to immunotherapy resistance in patients are the object of intense studies. Myeloid cells play a major role in tumors and include cells with different functions that can be grossly summarized as: (1) Antigen capture for presentation (dendritic cells, DCs) or for degradation (macrophages); (2) Tissue repair (macrophages) and (3) Effector function (mast cells, monocytes, granulocytes). However, the functional states of myeloid cells in human tumors are not completely understood. To study intact tumor microenvironments we have established a comprehensive approach for cellular and molecular analysis. Modular analysis of bulk RNAseq data of whole melanoma tissue sections stratified patient samples based on lymphocytic infiltrate but revealed inflammation and myeloid modules spread across samples. Polychromatic immunofluorescence and histocytometry showed that CD14+ cells represent the majority of the total tumor immune infiltrate. Furthermore, the majority of T cells present in melanoma tissue are in direct contact with CD14+ cells rather than melanoma cells. The distribution of CD14+ cells shows two distinct patterns: CD14+ cells within cancer nests (intratumoral) which are in close proximity to melanoma cells and are loaded with melanoma protein; CD14+ cells in the tissue surrounding cancer nests (stromal) which do not contain melanoma protein cargo and are organized in dense infiltrates. Using customized immunofluorescence-guided laser capture micro-dissection, we harvested CD14+ cells based on their tissue location and melanoma protein load for downstream analysis. We find that unsupervised clustering of transcriptomes groups cells by their tissue localization, i.e., intratumoral vs. stromal. Computational analysis, revealed distinct and replicable gene signatures associated with different pathways, in intratumoral and stromal CD14+ cells. Thus, the transcriptome differentiates the functional status of CD14+ cells related to their localization within tumor. Citation Format: Jan Martinek, Kyung In Kim, Te-Chia Wu, Victor Wang, Hannah M. Brookes, Lili Sun, Ananya Gulati, Joshy George, Philipp Henrich, Florentina Marches, Anthony Rongvaux, Michael Chiorazzi, Jeff Chuang, Paul Robson, Richard Flavell, Jacques Banchereau, Karolina Palucka. Transcriptional profiles of CD14+ cells in situ in melanoma tumors reveal plasticity and localization dependent function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1507.

Cell death is a common metazoan cell fate, and its inactivation is central to human malignancy. In Caenorhabditis elegans, apoptotic cell death occurs via the activation of the caspase CED-3 following binding of the EGL-1/BH3-only protein to the antiapoptotic CED-9/ BCL2 protein. Here we report a major alternative mechanism for caspase activation in vivo involving the F-box protein DRE-1. DRE-1 functions in parallel to EGL-1, requires CED-9 for activity, and binds to CED-9, suggesting that DRE-1 promotes apoptosis by inactivating CED-9. FBXO10, a human protein related to DRE-1, binds BCL2 and promotes its degradation, thereby initiating cell death. Moreover, some human diffuse large B-cell lymphomas have inactivating mutations in FBXO10 or express FBXO10 at low levels. Our results suggest that DRE-1/FBXO10 is a conserved regulator of apoptosis. Fil: Chiorazzi, Michael. The Rockefeller University. Laboratory of Developmental Genetics; Estados Unidos de América; Fil: Rui, Lixin. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Yang, Yandan. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Ceribelli, Michelle. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Tishbi, Nima. The Rockefeller University. Laboratory of Developmental Genetics; Estados Unidos de América; Fil: Maurer. Carine W.. The Rockefeller University. Laboratory of Developmental Genetics; Estados Unidos de América; Fil: Ranuncolo, Stella Maris. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina; National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina; Fil: Zhao, Hong. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Xu, Weihong. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Chan, Wing-Chung C.. University of Nebraska. Department of Pathology and Microbiology; Estados Unidos de América; Fil: Jaffe, Elaine S.. National Cancer Institute. Center for Cancer Research. Laboratory of Pathology; Estados Unidos de América; Fil: Gascoyne, Randy D.. British Columbia Cancer Agency; Canadá; Fil: Campo, Elias. Universidad de Barcelona. Hospital Clínico; España; Fil: Rossenwald, Andreas. University of Wurzburg. Department of Pathology; Alemania; Fil: Ott, German. Robert-Bosch-Krankenhaus and Dr. Margarete Fischer-Bosch Institute for Clinical Pharmacology. Department of Clinical Pathology; Alemania; Fil: Delabie, Jan. Oslo University Hospital. Pathology Clinic; Noruega; Fil: Rimsza, Lisa M.. University of Arizona. Department of Pathology; Estados Unidos de América; Southwest Oncology Group; Estados Unidos de América; Fil: Shaham, Shai. The Rockefeller University. Laboratory of Developmental Genetics; Estados Unidos de América; Fil: Staudt, Louis M.. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América

This article describes the legal research boot camp offered at the University of Arizona James E. Rogers College of Law. It is an intensive, one-week course offered the first week of the summer break to prepare students for their summer clerkships. It is also a vehicle to teach law librarianship students how to teach legal research.

In an attempt to write a history of legal research in law schools, Chiorazzi and Esposito found that very little has been written to chronicle the history of this important function of academic law libraries; but they did rediscover a gem written by Frederick Hicks, the godfather of law librarians, almost 100 years ago. Much of that which he speaks is still relevant. The authors decided that there was no better way to begin a discussion of the history of legal research instruction than the text of Hicks’ article. The authors have reprinted the entire Hicks article and have added their own comments throughout. And, of course, they knew no discussion of Hicks would be complete without the comments of Bob Berring on one of his personal hero's work. No law librarian has followed more closely in the intellectual footprints of Hicks than Professor Berring.

We used gene expression profiling to establish a molecular diagnosis of mantle cell lymphoma (MCL), to elucidate its pathogenesis, and to predict the length of survival of these patients. An MCL gene expression signature defined a large subset of MCLs that expressed cyclin D1 and a novel subset that lacked cyclin D1 expression. A precise measurement of tumor cell proliferation, provided by the expression of proliferation signature genes, identified patient subsets that differed by more than 5 years in median survival. Differences in cyclin D1 mRNA abundance synergized with INK4a/ARF locus deletions to dictate tumor proliferation rate and survival. We propose a quantitative model of the aberrant cell cycle regulation in MCL that provides a rationale for the design of cell cycle inhibitor therapy in this malignancy.

SUMMARY This article builds on Professor Penny Hazelton's examination of “how much of your collection is really available on Lexis and Westlaw?” While Hazelton examined the question from the perspective of space occupied, Professor Chiorazzi looks at the question from the perspective of collection use. Using the economic theory called the Pareto Principle, Chiorazzi argues that over 80% of the use of all legal materials is accounted for by the 20% of all legal materials that are available online. This has significant implications for law librarians seeking to expand or build new facilities. The legal research environment has changed to a point where most research can in fact be done online and large research paper collections may become harder to defend against administrators using potentially shortsighted cost-benefit analyses while making library budgeting decisions.

Circulating human B cells that coexpress V-preB and conventional L chains (V-preB+L+ B cells) are a recently described subset of B cells that express Abs with features of self-reactivity. Initial analysis of V-preB+L+ B cells was limited to Ig-κ and to the small, underused VH5 family. To determine whether Abs commonly expressed by V-preB+L+ B cells show similar features, we analyzed Ig H chains from three highly expressed VH families, VH1, VH3, and VH4, and Ig-λ. We find that VH1 and VH3 Abs expressed by V-preB+L+ B cells resemble VH5 in that they display increased JH6 use, long CDR3s, and an increased frequency of D-D fusions. Abs in all three of these VH families also show skewed D reading frame use resulting in predominance of hydrophobic amino acids, which are counterselected in conventional B cells. Like Ig-κ genes, the Ig-λ genes in V-preB+L+ B cells show long CDR3s, but they differ from Ig-κ genes in that they display no evidence of receptor editing. We conclude that a large number of H and L chain Abs expressed by V-preB+L+ B cells display features associated with self-reactive Abs.

Temporal control of programmed cell death is necessary to ensure that cells die at only the right time during animal development. How such temporal regulation is achieved remains poorly understood. In some Caenorhabditis elegans somatic cells, transcription of the egl-1/BH3-only gene promotes cell-specific death. The EGL-1 protein inhibits the CED-9/Bcl-2 protein, resulting in the release of the caspase activator CED-4/Apaf-1. Subsequent activation of the CED-3 caspase by CED-4 leads to cell death. Despite the important role of egl-1 transcription in promoting CED-3 activity in cells destined to die, it remains unclear whether the temporal control of cell death is mediated by egl-1 expression. Here, we show that egl-1 and ced-9 play only minor roles in the death of the C. elegans tail-spike cell, demonstrating that temporal control of tail-spike cell death can be achieved in the absence of egl-1. We go on to show that the timing of the onset of tail-spike cell death is controlled by transcriptional induction of the ced-3 caspase. We characterized the developmental expression pattern of ced-3, and show that, in the tail-spike cell, ced-3 expression is induced shortly before the cell dies, and this induction is sufficient to promote the demise of the cell. Both ced-3 expression and cell death are dependent on the transcription factor PAL-1, the C. elegans homolog of the mammalian tumor suppressor gene Cdx2. PAL-1 can bind to the ced-3promoter sites that are crucial for tail-spike cell death, suggesting that it promotes cell death by directly activating ced-3 transcription. Our results highlight a role that has not been described previously for the transcriptional regulation of caspases in controlling the timing of cell death onset during animal development.

A gene expression signature of tumor proliferation rate in mantle cell lymphoma (MCL) is an overriding molecular predictor of the length of survival following diagnosis. Many strongly proliferative MCL tumors have exceptionally high cyclin D1 mRNA levels and preferentially express short cyclin D1 mRNA isoforms. We demonstrate here that these short mRNAs are cyclin D1a isoforms with truncated 3′UTRs, not alternatively spliced cyclin D1b mRNA isoforms. Among 15 MCL tumors with truncated cyclin D1 mRNAs, 7 had genomic deletions in the CCND1 3′UTR region. In 3 others, CCND1 contained point mutations that created premature polyadenylation signals, giving rise to 1.5-kb mRNAs lacking most of the 3′UTR. Both types of genomic alteration created transcripts lacking mRNA destabilization elements present in the wild-type cyclin D1a mRNA. Premature polyadenylation due to a 3′UTR mutation also was present in the Z-138 MCL cell line, which expressed both truncated and full-length cyclin D1a mRNAs. In these cells, the half-life of the short cyclin D1a mRNA was much longer than that of the full-length mRNA. We conclude that alterations of CCND1 3′UTR structure can significantly increase its oncogenic effect and worsen the clinical course of MCL patients.

Cyclin D1 overexpression is believed to be essential in the pathogenesis of mantle cell lymphoma (MCL). Hence, the existence of cyclin D1-negative MCL has been controversial and difficult to substantiate. Our previous gene expression profiling study identified several cases that lacked cyclin D1 expression, but had a gene expression signature typical of MCL. Herein, we report the clinical, pathologic, and genetic features of 6 cases of cyclin D1-negative MCL. All 6 cases exhibited the characteristic morphologic features and the unique gene expression signature of MCL but lacked the t(11;14)(q13; q32) by fluorescence in situ hybridization (FISH) analysis. The tumor cells also failed to express cyclin D1 protein, but instead expressed either cyclin D2 (2 cases) or cyclin D3 (4 cases). There was good correlation between cyclin D protein expression and the corresponding mRNA expression levels by gene expression analysis. Using interphase FISH, we did not detect chromosomal translocations or amplifications involving CCND2 and CCND3 loci in these cases. Patients with cyclin D1-negative MCL were similar clinically to those with cyclin D1-positive MCL. In conclusion, cases of cyclin D1-negative MCL do exist and are part of the spectrum of MCL. Up-regulation of cyclin D2 or D3 may substitute for cyclin D1 in the pathogenesis of MCL.

background Patients with follicular lymphoma may survive for periods of less than 1 year to more than 20 years after diagnosis. We used gene-expression profiles of tumor-biopsy specimens obtained at diagnosis to develop a molecular predictor of the length of survival. methods Gene-expression profiling was performed on 191 biopsy specimens obtained from patients with untreated follicular lymphoma. Supervised methods were used to discover expression patterns associated with the length of survival in a training set of 95 specimens. A molecular predictor of survival was constructed from these genes and validated in an independent test set of 96 specimens. results Individual genes that predicted the length of survival were grouped into gene-expression signatures on the basis of their expression in the training set, and two such signatures were used to construct a survival predictor. The two signatures allowed patients with specimens in the test set to be divided into four quartiles with widely disparate median lengths of survival (13.6, 11.1, 10.8, and 3.9 years), independently of clinical prognostic variables. Flow cytometry showed that these signatures reflected gene expression by nonmalignant tumor-infiltrating immune cells. conclusions The length of survival among patients with follicular lymphoma correlates with the molecular features of nonmalignant immune cells present in the tumor at diagnosis.

B cells develop from hematopoietic stem cells (HSC) in the bone marrow (BM) through a number of distinct stages before they migrate to the periphery as naïve mature B lymphocytes. These developmental stages can be identified by expression of cell surface antigens and Ig gene rearrangement status. The aim of this study was to characterize the earliest steps of normal human B cell development by gene expression profiling. Immunomagnetic selection and subsequent fluorescence-activated cell sorting (FACS) were used to isolate five populations from adult human BM: CD34+CD38− (HSC), CD34+CD10+CD19− early lymphoid progenitor cells (ELP), CD34+CD10+CD19+IgM− progenitor B cells (pro-B), CD34−CD10+CD19+IgM− precursor B cells (pre-B) and CD34−CD10+CD19+IgM+ immature B cells (IM). Total RNA was extracted from the purified cell populations, amplified and hybridized to Lymphochip cDNA microarrays. Six independent experiments from different donors were performed for each cell population. Expression of the genes encoding the selection markers confirmed the validity of the approach. Interestingly, genes necessary for the V(D)J-recombination such as RAG-1, RAG-2, TdT and ADA showed higher gene expression in the ELP population than in the HSC. In contrast, the transcription factors E2A, EBF, and Pax-5, which are all essential for early B-cell development, were first turned on in pro-B cells, in accordance with the B-cell lineage commitment. The ELP did not express B, T or NK lineage markers, except for a higher expression level of CD2 in the ELP population than in the four other cell populations. Taken together, the expression pattern of CD2 and the V(D)J-recombination genes in the ELP population, indicate that these cells have developed a lymphocyte potential, but are not fully committed to B-lineage cells. Hierarchical cluster analysis of the 758 differentially expressed genes (differences in relative expression by a factor of two or more and with maximum10% FDR) revealed a pattern that clearly separated the five consecutive cell populations. Furthermore, we created expression signatures based on information from Gene Ontology (GO) http://source.stanford.edu/cgi-bin/source/sourceSearch. One of the clearest distinctions between the gene expressions of the five developmental populations involved genes associated with proliferation, and showed that the HSC and IM populations are relatively indolent while the pro-B and pre-B populations comprised high expression levels of nearly all the proliferation associated genes. Finally, we examined in further detail the transitions between HSC, ELP and pro B cells. We found 25 genes to be differently expressed in the ELP population in comparison to the HSC and pro-B populations, including IGJ, BCL2 and BLNK. To identify combinations of markers that could better discriminate the ELP population, we also performed a gene pair class separation test. This resulted in 68 gene pairs with score above 10 that were denoted very good discriminators. For several of the markers the differences in gene expression were verified at the protein level by five colour FACS analysis. Taken together, these results provide new insight into the molecular processes that take place in the early human B cell differentiation, and in particular provide new information regarding expression of genes in the ELP population.

Clinical management differs significantly for the various types of non-Hodgkin lymphoma (NHL), and the diagnosis of these lymphomas can be challenging in some cases. Further, existing NHL categories include subgroups that can differ substantially in gene expression, response to therapy and overall survival. We have created a custom oligonucleotide microarray, named LymphDx, which could prove clinically useful for molecular diagnosis and outcome prediction in NHL. Biopsy specimens were obtained from 559 patients with a variety of lymphomas and lymphoproliferative conditions. Gene expression profiles of these samples were obtained using Affymetrix U133 A and B microarrays. The 2653 genes on LymphDx were chosen to include:(1)Genes most differentially expressed among NHL types based on Affymetrix U133 or Lymphochip microarrays (2)Genes predicting length of survival in diffuse large B cell lymphoma(DLBCL), follicular lymphoma(FL) and mantle cell lymphoma(MCL) (3)Genes encoded in the EBV and HHV-8 viral genomes (4)Genes encoding all known surface markers, kinases, cytokines and their receptors, as well as oncogenes, tumor suppressors, and other genes relevant to lymphoma. The LymphDx microarray was used to profile gene expression in 434 biopsy samples. These data were used to create a diagnostic algorithm that can distinguish various NHL types and benign follicular hyperplasia(FH) based on gene expression. The algorithm classifies a sample into one of the following categories: Burkitt’s lymphoma(BL), DLBCL, FL, MCL, small lymphocytic lymphoma(SLL) or FH. The algorithm further distinguishes the 3 recognized DLBCL subgroups: germinal center B cell-like, activated B cell-like or primary mediastinal lymphoma. Using a leave one out, cross validation strategy, the algorithm was found to agree well with the pathology diagnosis (see Figure). Some samples were deemed unclassified when their gene expression did not adequately match with that of any of the NHL categories. For a few samples, the gene expression-based diagnosis and the pathology diagnosis were discordant. Pathology review showed that two NHL types coexisted (eg FL and DLBCL) in many of these cases, potentially explaining the results of the diagnostic algorithm. LymphDx could also reliably predict the overall survival of patients with DLBCL, FL and MCL. Prospective evaluation of the LymphDx microarray is warranted since it could be used to provide objective molecular diagnostic, and prognostic information for patients with NHL. Figure Figure

Most MCLs harbor the t(11;14) translocation resulting in overexpression of cyclin D1 that promotes tumor proliferation. Gene expression profiling of 92 cyclin D1 positive MCL biopsy samples defined a gene expression signature of tumor proliferation that provided strong prognostic information: the quartile of patients with the highest proliferation gene expression signature had a 0.8 year median survival whereas the quartile with the lowest expression had a 6.7 year median survival (Cancer Cell 2003, 3:185). Sixteen of the 23 MCL samples in the highest proliferation quartile displayed exceptionally high cyclin D1 levels and preferentially expressed short cyclin D1 mRNA forms that lacked parts of the 3′UTR known to harbor RNA-destabilizing elements. To test whether expression of short cyclin D1 mRNA is due to genomic deletions, we screened DNA from all samples in the highest proliferation quartile by quantitative genomic PCR. We found genomic deletions of the 3′UTR region in 6 of the 10 samples that expressed short mRNAs. We next sequenced the proximal 400bp region of the 3′UTR in those samples with high expression of short mRNAs that did not have genomic deletions. In 2 samples, we found 2 distinct mutations: a 3bp deletion and an A insertion. Both of these mutations created a premature polyadenylation signal (AATAAA) about 300bp 3′ of the stop codon, giving rise to a predicted mRNA of 1.5kb that lacks the destabilizing elements present in the full-length form. We identified two MCL cell lines, Jeko-1 and Z138, that also showed high-level expression of the short cyclin D1 form. In Jeko-1 we found a deletion of the 3′UTR similar to that observed in the MCL patient samples, whereas the mechanism responsible for expression of the short cyclin D1 mRNA in Z138 remains unclear. In both lines, we confirmed that the half-life of the short cyclin D1 mRNA is longer than that of the full length mRNA. Our results demonstrate that MCLs increase cyclin D1 expression not only through the t(11;14), but also by the additional mechanisms of genomic deletions and mutations that create alternative polyadenylation sites that yield more stable mRNAs. Higher cyclin D1 expression increases proliferation and decreases survival, suggesting that strategies aimed at reducing cyclin D1 expression in MCL could have therapeutic promise.