HTS384 NCI60: The Next Phase of the NCI60 Screen.

The NCI60 human tumor cell line screen has been in operation as a service to the cancer research community for over 30 years. The screen operated with 96-well plates, a 2-day exposure period to test agents, and, following cell fixation, a visible absorbance endpoint by the protein-staining dye sulforhodamine B. Here, we describe the next phase of this important cancer research tool, the HTS384 NCI60 screen. While the cell lines remain the same, the updated screen is performed with 384-well plates, a 3-day exposure period to test agents, and a luminescent endpoint to measure cell viability based upon cellular ATP content. In this study, a library of 1003 FDA-approved and investigational small molecule anticancer agents was screened by the two NCI60 assays. The datasets were compared with a focus on targeted agents with at least six representatives in the library. For many agents, including inhibitors of EGFR, BRAF, MEK, ERK, and PI3K, the patterns of GI50 values were very similar between the screens with strong correlations between those patterns within the dataset from each screen. However, for some groups of targeted agents, including mTOR, BET bromodomain, and NAMPRTase inhibitors, there were limited or no correlations between the two datasets, although the patterns of GI50 values and correlations between those patterns within each dataset were apparent. Beginning in January 2024, the HTS384 NCI60 screen became the free screening service of the National Cancer Institute to facilitate drug discovery by the cancer research community.

Antibody drug conjugate adverse effects can be understood and addressed based on immune complex clearance mechanisms.

Numerous antibody-drug conjugates (ADC) are being developed for cancer immunotherapy. Although several of these agents have demonstrated considerable clinical efficacy and have won FDA approval, in many instances they have been characterized by adverse side effects (ASE) which can be quite severe in a fraction of treated patients. The key hypothesis in this perspective is that many of the most serious ASE associated with the use of ADC in the treatment of cancer can be most readily explained and understood due to the inappropriate processing of these ADC via pathways normally followed for immune complex clearance, which include phagocytosis and trogocytosis. We review the key published basic science experiments and clinical observations that support this idea. We propose that it is the interaction of the ADC with Fc receptors expressed on off-target cells and tissues that can most readily explain ADC-mediated pathologies which therefore provides a rationale for the design of protocols to minimize ASE. We describe measurements that should help to identify those patients most likely to experience ASE due to ADC and we propose readily available treatments as well as therapies under development for other indications that should substantially reduce ASE associated with ADC. Our focus will be on the following FDA-approved ADC for which there are substantial literatures: gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin; and trastuzumab emtansine (T-DM1), and trastuzumab deruxtecan (T-DXd).

Oligosaccharide production and signaling correlate with delayed flowering in an Arabidopsis genotype grown and selected in high [CO2].

Since industrialization began, atmospheric CO2 ([CO2]) has increased from 270 to 415 ppm and is projected to reach 800-1000 ppm this century. Some Arabidopsis thaliana (Arabidopsis) genotypes delayed flowering in elevated [CO2] relative to current [CO2], while others showed no change or accelerations. To predict genotype-specific flowering behaviors, we must understand the mechanisms driving flowering response to rising [CO2]. [CO2] changes alter photosynthesis and carbohydrates in plants. Plants sense carbohydrate levels, and exogenous carbohydrate application influences flowering time and flowering transcript levels. We asked how organismal changes in carbohydrates and transcription correlate with changes in flowering time under elevated [CO2]. We used a genotype (SG) of Arabidopsis that was selected for high fitness at elevated [CO2] (700 ppm). SG delays flowering under elevated [CO2] (700 ppm) relative to current [CO2] (400 ppm). We compared SG to a closely related control genotype (CG) that shows no [CO2]-induced flowering change. We compared metabolomic and transcriptomic profiles in these genotypes at current and elevated [CO2] to assess correlations with flowering in these conditions. While both genotypes altered carbohydrates in response to elevated [CO2], SG had higher levels of sucrose than CG and showed a stronger increase in glucose and fructose in elevated [CO2]. Both genotypes demonstrated transcriptional changes, with CG increasing genes related to fructose 1,6-bisphosphate breakdown, amino acid synthesis, and secondary metabolites; and SG decreasing genes related to starch and sugar metabolism, but increasing genes involved in oligosaccharide production and sugar modifications. Genes associated with flowering regulation within the photoperiod, vernalization, and meristem identity pathways were altered in these genotypes. Elevated [CO2] may alter carbohydrates to influence transcription in both genotypes and delayed flowering in SG. Changes in the oligosaccharide pool may contribute to delayed flowering in SG. This work extends the literature exploring genotypic-specific flowering responses to elevated [CO2].

Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls.

Trogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb-based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA-mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell-bound cognate IgG mAbs for several different mAb-donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP-1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody-drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trogocytosis of mAb-opsonized donor cells mediated by adherent THP-1 cells Alternate Protocol: Application of fluorescence microscopy to examine THP-1 cell-mediated trogocytosis Support Protocol 1: Alexa labeling of mAbs and determination of F/P ratios Support Protocol 2: Standard washing procedure Support Protocol 3: Labeling and opsonization of cells Basic Protocol 2: Trogocytosis mediated by human monocytes as acceptor cells Support Protocol 4: Isolation of human monocytes Basic Protocol 3: Trogocytosis mediated by THP-1 cells in solution Support Protocol 5: Retinoic acid treatment of THP-1 cells Support Protocol 6: Culturing of SCC-25, BT-474, MOLT-4 and THP-1 cell lines.

Cold exposure induces vaso-occlusion and pain in sickle mice that depend on complement activation.

Vaso-occlusive pain episodes (VOE) cause severe pain in patients with sickle cell disease (SCD). Vaso-occlusive events promote ischemia/reperfusion pathobiology that activates complement. We hypothesized that complement activation is linked to VOE. We used cold to induce VOE in the Townes sickle homozygous for hemoglobin S (HbSS) mouse model and complement inhibitors to determine whether anaphylatoxin C5a mediates VOE. We used a dorsal skinfold chamber to measure microvascular stasis (vaso-occlusion) and von Frey filaments applied to the plantar surface of the hind paw to assess mechanical hyperalgesia in HbSS and control Townes mice homozygous for hemoglobin A (HbAA) mice after cold exposure at 10°C/50°F for 1 hour. Cold exposure induced more vaso-occlusion in nonhyperalgesic HbSS mice (33%) than in HbAA mice (11%) or HbSS mice left at room temperature (1%). Cold exposure also produced mechanical hyperalgesia as measured by paw withdrawal threshold in HbSS mice compared with that in HbAA mice or HbSS mice left at room temperature. Vaso-occlusion and hyperalgesia were associated with an increase in complement activation fragments Bb and C5a in plasma of HbSS mice after cold exposure. This was accompanied by an increase in proinflammatory NF-κB activation and VCAM-1 and ICAM-1 expression in the liver. Pretreatment of nonhyperalgesic HbSS mice before cold exposure with anti-C5 or anti-C5aR monoclonal antibodies (mAbs) decreased vaso-occlusion, mechanical hyperalgesia, complement activation, and liver inflammatory markers compared with pretreatment with control mAb. Anti-C5 or -C5aR mAb infusion also abrogated mechanical hyperalgesia in HbSS mice with ongoing hyperalgesia at baseline. These findings suggest that C5a promotes vaso-occlusion, pain, and inflammation during VOE and may play a role in chronic pain.

Rituximab induced cytokine release with high serum IP-10 (CXCL10) concentrations is associated with infusion reactions.

Monoclonal antibody induced infusion reactions (IRs) can be serious and even fatal. We used clinical data and blood samples from 37 treatment naïve patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) initiating therapy for progressive disease with a single 50 mg dose of intravenous (IV) rituximab at 25 mg/h. Twenty-four (65 %) patients had IRs at a median of 78 min (range 35-128) and rituximab dose of 32 mg (range 15-50). IR risk did not correlate with patient or CLL characteristics, CLL counts or CD20 levels, or serum rituximab or complement concentrations. Thirty-five (95 %) patients had cytokine release response with a ≥ 4-fold increase in serum concentration of ≥ 1 inflammatory cytokine. IRs were associated with significantly higher post-infusion serum concentrations of gamma interferon induced cytokines IP-10, IL-6 and IL-8. IP-10 concentrations increased ≥ 4-fold in all patients with an IR and were above the upper limit of detection (40,000 pg/ml) in 17 (71 %). In contrast, to only three (23 %) patients without an IR had an ≥ 4-fold increase in serum concentrations of IP-10 (highest 22,013 pg/ml). Our data suggest that cytokine release could be initiated by activation of effector cells responsible for clearance of circulating CLL cells with IRs occurring in those with higher levels of gamma interferon induced cytokines. These novel insights could inform future research to better understand and manage IRs and understand the role of cytokines in the control of cytotoxic immune responses to mAb.

Phagocytosis via complement receptor 3 enables microbes to evade killing by neutrophils.

CR3 (CD11b/CD18; αmß2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species, and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae that does not express phase-variable Opa proteins suppresses neutrophil reactive oxygen species and delays phagolysosome formation. Here, binding and internalization of Opa-deleted (Δopa) N. gonorrhoeae by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. No detectable C3 was deposited on N. gonorrhoeae in the presence of neutrophils alone. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced Δopa N. gonorrhoeae phagocytosis, which required the CD11b I-domain. Phagocytosis of N. gonorrhoeae was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of Δopa N. gonorrhoeae. Neutrophils exposed to Δopa N. gonorrhoeae had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis, which also resides in immature phagosomes, was CR3-dependent and did not elicit reactive oxygen species. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing.

A cloud-based resource for genome coordinate-based exploration and large-scale analysis of chromosome aberrations and gene fusions in cancer.

Cytogenetic analysis provides important information on the genetic mechanisms of cancer. The Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (Mitelman DB) is the largest catalog of acquired chromosome aberrations, presently comprising >70 000 cases across multiple cancer types. Although this resource has enabled the identification of chromosome abnormalities leading to specific cancers and cancer mechanisms, a large-scale, systematic analysis of these aberrations and their downstream implications has been difficult due to the lack of a standard, automated mapping from aberrations to genomic coordinates. We previously introduced CytoConverter as a tool that automates such conversions. CytoConverter has now been updated with improved interpretation of karyotypes and has been integrated with the Mitelman DB, providing a comprehensive mapping of the 70 000+ cases to genomic coordinates, as well as visualization of the frequencies of chromosomal gains and losses. Importantly, all CytoConverter-generated genomic coordinates are publicly available in Google BigQuery, a cloud-based data warehouse, facilitating data exploration and integration with other datasets hosted by the Institute for Systems Biology Cancer Gateway in the Cloud (ISB-CGC) Resource. We demonstrate the use of BigQuery for integrative analysis of Mitelman DB with other cancer datasets, including a comparison of the frequency of imbalances identified in Mitelman DB cases with those found in The Cancer Genome Atlas (TCGA) copy number datasets. This solution provides opportunities to leverage the power of cloud computing for low-cost, scalable, and integrated analysis of chromosome aberrations and gene fusions in cancer.

FcγR-Mediated Trogocytosis 2.0: Revisiting History Gives Rise to a Unifying Hypothesis.

There is increasing interest in the clinical implications and immunology of trogocytosis, a process in which the receptors on acceptor cells remove and internalize cognate ligands from donor cells. We have reported that this phenomenon occurs in cancer immunotherapy, in which cells that express FcγR remove and internalize CD20 and bound mAbs from malignant B cells. This process can be generalized to include other reactions including the immune adherence phenomenon and antibody-induced immunosuppression. We discuss in detail FcγR-mediated trogocytosis and the evidence supporting a proposed predominant role for liver sinusoidal endothelial cells via the action of the inhibitory receptor FcγRIIb2. We describe experiments to test the validity of this hypothesis. The elucidation of the details of FcγR-mediated trogocytosis has the potential to allow for the development of novel therapies that can potentially block or enhance this reaction, depending upon whether the process leads to unfavorable or positive biological effects.

Folic acid-mediated fibrosis is driven by C5a receptor 1-mediated activation of kidney myeloid cells.

We have previously reported that increased expression and activation of kidney cell complement components play an important role in the pathogenesis of renal scarring. Here, we used floxed green fluorescent protein (GFP)-C5a receptor 1 (C5aR1) knockin mice (GFP-C5ar1fl/fl) and the model of folic acid (FA)-induced kidney injury to define the cell types and potential mechanisms by which increased C5aR1 activation leads to fibrosis. Using flow cytometry and confocal microscopy, we identified macrophages as the major interstitial cell type showing increased expression of C5aR1 in FA-treated mice. C5ar1fl/fl.Lyz2Cre+/- mice, in which C5aR1 has been specifically deleted in lysozyme M-expressing myeloid cells, experienced reduced fibrosis compared with control C5ar1fl/fl mice. Examination of C5aR1-expressing macrophage transcriptomes by gene set enrichment analysis demonstrated that these cells were enriched in pathways corresponding to the complement cascade, collagen formation, and the NABA matrisome, strongly pointing to their critical roles in tissue repair/scarring. Since C5aR1 was also detected in a small population of platelet-derived growth factor receptor-ß+ GFP+ cells, we developed C5ar1fl/fl.Foxd1Cre+/- mice, in which C5aR1 is deleted specifically in pericytes, and found reduced FA-induced fibrosis. Primary cell cultures of platelet-derived growth factor receptor-ß+ pericytes isolated from FA-treated C5ar1fl/fl.Foxd1Cre+/- mice showed reduced secretion of several cytokines, including IL-6 and macrophage inflammatory protein-2, compared with pericytes isolated from FA-treated control GFP-C5ar1fl/fl mice. Collectively, these data imply that C5a/C5aR1 axis activation primarily in interstitial cells contributes to the development of renal fibrosis.NEW & NOTEWORTHY This study used novel green fluorescent protein C5a receptor 1 floxed mice and the model of folic acid-mediated kidney fibrosis to demonstrate the pathogenic role of increased expression of this complement receptor on macrophages.

Special Issue: The Role of Complement in Cancer Immunotherapy.

The complement system plays an important role in critical aspects of immune defense and in the maintenance of homeostasis in the bloodstream, as well as in essentially all tissues and organs [...].

Risk-adapted, ofatumumab-based chemoimmunotherapy and consolidation in treatment-naïve chronic lymphocytic leukemia: a phase 2 study.

High-risk cytogenetics and minimal residual disease (MRD) after chemoimmunotherapy (CIT) predict unfavorable outcome in chronic lymphocytic leukemia (CLL). This phase 2 study investigated risk-adapted CIT in treatment-naïve CLL (NCT01145209). Patients with high-risk cytogenetics received induction with fludarabine, cyclophosphamide, and ofatumumab. Those without high-risk cytogenetics received fludarabine and ofatumumab. After induction, MRD positive (MRD+) patients received 4 doses of ofatumumab consolidation. MRD negative (MRD-) patients had no intervention. Of 28 evaluable for response, all responded to induction and 10 (36%) achieved MRD-. Two-year progression-free survival (PFS) was 71.4% (CI95, 56.5-90.3%). There was no significant difference in median PFS between the high-risk and the standard-risk groups. Ofatumumab consolidation didn't convert MRD + to MRD-. In the MRD + group, we saw selective loss of CD20 antigens during therapy. In conclusion, risk-adapted CIT is feasible in treatment-naïve CLL. Ofatumumab consolidation didn't improve depth of response in MRD + patients. Loss of targetable CD20 likely reduces efficacy of consolidation therapy.

The Role of Complement in the Mechanism of Action of Therapeutic Anti-Cancer mAbs.

Unconjugated anti-cancer IgG1 monoclonal antibodies (mAbs) activate antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells and antibody-dependent cellular phagocytosis (ADCP) by macrophages, and these activities are thought to be important mechanisms of action for many of these mAbs in vivo. Several mAbs also activate the classical complement pathway and promote complement-dependent cytotoxicity (CDC), although with very different levels of efficacy, depending on the mAb, the target antigen, and the tumor type. Recent studies have unraveled the various structural factors that define why some IgG1 mAbs are strong mediators of CDC, whereas others are not. The role of complement activation and membrane inhibitors expressed by tumor cells, most notably CD55 and CD59, has also been quite extensively studied, but how much these affect the resistance of tumors in vivo to IgG1 therapeutic mAbs still remains incompletely understood. Recent studies have demonstrated that complement activation has multiple effects beyond target cell lysis, affecting both innate and adaptive immunity mediated by soluble complement fragments, such as C3a and C5a, and by stimulating complement receptors expressed by immune cells, including NK cells, neutrophils, macrophages, T cells, and dendritic cells. Complement activation can enhance ADCC and ADCP and may contribute to the vaccine effect of mAbs. These different aspects of complement are also briefly reviewed in the specific context of FDA-approved therapeutic anti-cancer IgG1 mAbs.

Clearance of amyloid-beta with bispecific antibody constructs bound to erythrocytes.

We propose use of bispecific monoclonal antibody (mAb) complexes bound to erythrocytes to redress the lack of efficacy of anti-amyloid beta mAbs in Alzheimer's disease treatment. Our paradigm leverages erythrocyte complement receptor 1 to promote rapid and quantitative removal of amyloid beta from the circulation, and its subsequent removal from the brain as well.

How Do mAbs Make Use of Complement to Kill Cancer Cells? The Role of Ca2.

We examined the kinetics and mechanisms by which monoclonal antibodies (mAbs) utilize complement to rapidly kill targeted cancer cells. Based on results from flow cytometry, confocal microscopy and high-resolution digital imaging experiments, the general patterns which have emerged reveal cytotoxic activities mediated by substantial and lethal Ca2+ fluxes. The Ca2+ fluxes are common to the reported pathways that have been utilized by other toxins in killing nucleated cells. These reactions terminate in very high levels of cell killing, and based on these considerations, we suggest additional strategies to further enhance mAb-based targeting of cancer with complement.

Mutation of complement factor B causing massive fluid-phase dysregulation of the alternative complement pathway can result in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome is an ultra-rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute kidney injury. Its pathogenesis is driven most frequently by dysregulated cell-surface control of the alternative pathway of complement secondary to inherited and/or acquired factors. Here we evaluated two unrelated patients with atypical hemolytic uremic syndrome. The first, a five-year-old Caucasian female, presented at 10 months with schistocytes, thrombocytopenia and kidney injury. The second, a 55-year-old Caucasian female, presented at age 31 following caesarean section for preeclampsia. Complement biomarker testing was remarkable for undetectable levels of C3 in both. Circulating levels of C5 and properdin were also low consistent with over-activity of the alternative and terminal pathways of complement. Genetic testing identified a heterozygous novel variant in CFB (c.1101 C>A, p.Ser367Arg) in both patients. Functional studies found strong fluid-phase C3 cleavage when normal and proband sera were mixed. Cell-surface C3b deposition was strongly positive when patient serum was supplemented with C3. In vitro control of C3 convertase activity could be restored with increased concentrations of factor H. Thus, CFB p.Ser367Arg is a gain-of-function pathogenic variant that leads to dysregulation of the alternative pathway in the fluid-phase and increased C3b deposition on cell surfaces. Our study highlights the complexities of complement-mediated diseases like atypical hemolytic uremic syndrome and illustrates the importance of functional studies at the variant level to gain insight into the disease phenotype.

C3(H2O) prevents rescue of complement-mediated C3 glomerulopathy in Cfh-/- Cfd-/- mice.

Therapeutic complement inhibition is a major focus for novel drug development. Of upstream targets, factor D (FD) is appealing because it circulates in plasma at low concentrations and has a single function: to cleave factor B to generate C3 convertase of the alternative pathway (AP). Mice with a targeted deletion of factor H (FH; Cfh-/- mice) develop C3 glomerulopathy (C3G) due to uncontrolled AP activity. To assess the impact of FD inhibition, we studied Cfh-/- Cfd-/- mice. We show that C3G in Cfh-/- mice is not rescued by removing FD. We used serum from Cfh-/- Cfd-/- mice to demonstrate that residual AP function occurs even when both FD and FH are missing and that hemolytic activity is present due to the action of C3(H2O). We propose that uncontrolled tick-over leads to slow activation of the AP in Cfh-/- Cfd-/- mice and that a minimal threshold of FH is necessary if tissue deposition of C3 is to be prevented. The FD/FH ratio dictates serum C3 level and renal C3b deposition. In C3G patients with chronic renal disease, the FD/FH ratio correlates inversely with C3 and C5 serum levels, suggesting that continuous AP control may be difficult to achieve by targeting FD.

DuoHexaBody-CD37®, a novel biparatopic CD37 antibody with enhanced Fc-mediated hexamerization as a potential therapy for B-cell malignancies.

Tetraspanin CD37 has recently received renewed interest as a therapeutic target for B-cell malignancies. Although complement-dependent cytotoxicity (CDC) is a powerful Fc-mediated effector function for killing hematological cancer cells, CD37-specific antibodies are generally poor inducers of CDC. To enhance CDC, the E430G mutation was introduced into humanized CD37 monoclonal IgG1 antibodies to drive more efficient IgG hexamer formation through intermolecular Fc-Fc interactions after cell surface antigen binding. DuoHexaBody-CD37, a bispecific CD37 antibody with the E430G hexamerization-enhancing mutation targeting two non-overlapping epitopes on CD37 (biparatopic), demonstrated potent and superior CDC activity compared to other CD37 antibody variants evaluated, in particular ex vivo in patient-derived chronic lymphocytic leukemia cells. The superior CDC potency was attributed to enhanced IgG hexamerization mediated by the E430G mutation in combination with dual epitope targeting. The mechanism of action of DuoHexaBody-CD37 was shown to be multifaceted, as it was additionally capable of inducing efficient antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vitro. Finally, potent anti-tumor activity in vivo was observed in cell line- and patient-derived xenograft models from different B-cell malignancy subtypes. These encouraging preclinical results suggest that DuoHexaBody-CD37 (GEN3009) may serve as a potential therapeutic antibody for the treatment of human B-cell malignancies.

Author Correction: 'Stealth' corporate innovation: an emerging threat for therapeutic drug development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.