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In recent years, the rapid advancement of generative artificial intelligence (GenAI) has revolutionized the landscape of drug design, offering innovative solutions to potentially expedite the discovery of novel therapeutics. GenAI encompasses algorithms and models that autonomously create new data, including text, images, and molecules, often mirroring characteristics of existing datasets. This comprehensive review delves into the realm of GenAI for drug design, emphasizing recent advancements and methodologies that have propelled the field forward. Specifically, we focus on three prominent paradigms: transformers, diffusion models, and reinforcement learning algorithms, which have been exceptionally impactful in the last few years. By synthesizing insights from a myriad of studies and developments, we elucidate the potential of these approaches in accelerating the drug discovery process. Through a detailed analysis, we explore the current state and future directions of GenAI in the context of drug design, highlighting its transformative impact on pharmaceutical research and development., (Copyright © 2024. Published by Elsevier Ltd.)

During the past decade promising methods for computational prediction of electron ionization mass spectra have been developed. The most prominent ones are based on quantum chemistry (QCEIMS) and machine learning (CFM-EI, NEIMS). Here we provide a threefold comparison of these methods with respect to spectral prediction and compound identification. We found that there is no unambiguous way to determine the best of these three methods. Among other factors, we find that the choice of spectral distance functions play an important role regarding the performance for compound identification.

Diffuse large B-cell lymphoma featuring overexpression of MYC and B-Cell Lymphoma 2 (double expressor lymphoma, DEL) is associated with poor outcomes. Existing evidence suggesting improved outcomes for DEL with the use of more intensive regimens than R-CHOP is restricted to younger patients and based on limited evidence from low patient numbers. We retrospectively evaluated the impact of intensive frontline regimens versus R-CHOP in a multicenter analysis across 7 academic medical centers in the United States. We collected 90 cases of DEL, 46 out of 90 patients (51%) received R-CHOP and 44/90 (49%) received an intensive regimen, which was predominantly DA-EPOCH-R. Treatment cohorts were evenly balanced for demographics and disease characteristics, though the intensive group had a higher lactate dehydrogenase (LDH, 326 vs. 230 U/L p = 0.06) and presence of B-symptoms (50% vs. 22%, p = 0.01) compared to the R-CHOP cohort. There was no difference in PFS (median 53 vs. 38 months, p = 0.49) or overall survival (67 vs. not reached months, p = 0.14) between the R-CHOP and intensive therapy cohorts, respectively. On multivariate analysis, intensive therapy was associated with a hazard ratio of 2.35 (95% CI 0.74-7.41), though this was not statistically significant. Additionally, a subgroup analysis of intermediate high-risk lymphoma defined by IPI ≥3 did not identify a difference in survival outcomes between regimens. We conclude that in our multi-center cohort there is no evidence supporting the use of intensive regimens over R-CHOP, suggesting that R-CHOP remains the standard of care for treating DEL., (© 2021 John Wiley & Sons Ltd.)

Background: All-trans retinoic acid (ATRA) serves as the backbone of the management of patients with acute promyelocytic leukemia (APL), with guidelines recommending the initiation of ATRA as soon as APL is suspected. As a regional referral center for patients with acute leukemia, those who are suspected of having APL are often transferred to our facility. However, many referring centers are unable to initiate treatment using ATRA. We conducted an exploratory analysis of the clinical availability of ATRA and the factors limiting access to this critical drug., Patients and Methods: The United States was divided into 6 geographic regions: Northwest, Southwest, Central, Southeast, Northeast, and the Great Lakes. Twenty hospitals were randomly selected from states within each of these regions and were surveyed as to whether they typically treated patients with acute leukemia, the availability of ATRA at their institution, and reported reasons for not stocking ATRA (if not available)., Results: Less than one-third of hospitals queried (31%) had ATRA in stock. Neither the size of the hospital nor the hospital's status as academic versus nonacademic (53% vs 31%; P=.08) influenced ATRA availability. Of the hospitals that referred patients with APL, only 14% (7/49) had ATRA readily available. Hospitals that treated patients with APL were more likely to have ATRA available than referring centers (58% vs 14%; P=.000002)., Conclusions: Nearly two-thirds of the hospitals surveyed that cared for patients with acute leukemia do not have ATRA immediately available. Moreover, the vast majority of hospitals that refer patients to other centers do not have ATRA. These findings should spur investigation into the impact of immediate ATRA availability on the morbidity and mortality of patients with APL. A call by hematologists nationwide to their formulary committees is warranted to ensure that this lifesaving medication is available to patients suspected of having APL.

Purpose: On the basis of the recent discovery of mutations in Bruton tyrosine kinase ( BTK ) in follicular lymphoma, we studied their functional properties., Experimental Design: We identified novel somatic BTK mutations in 7% of a combined total of 139 follicular lymphoma and 11 transformed follicular lymphoma cases, none of which had received prior treatment with B-cell receptor (BCR) targeted drugs. We reconstituted wild-type (WT) and mutant BTK into various engineered lymphoma cell lines. We measured BCR-induced signal transduction events in engineered cell lines and primary human follicular lymphoma B cells., Results: We uncovered that all BTK mutants destabilized the BTK protein and some created BTK kinase-dead mutants. The phospholipase C gamma 2 (PLCγ2) is a substrate of BTK but the BTK mutants did not alter PLCγ2 phosphorylation. Instead, we discovered that BTK mutants induced an exaggerated AKT phosphorylation phenotype in anti-Ig-treated recombinant lymphoma cell lines. The short hairpin RNA-mediated knockdown of BTK expression in primary human nonmalignant lymph node-derived B cells resulted in strong anti-Ig-induced AKT activation, as did the degradation of BTK protein in cell lines using ibrutinib-based proteolysis targeting chimera. Finally, through analyses of primary human follicular lymphoma B cells carrying WT or mutant BTK , we detected elevated AKT phosphorylation following surface Ig crosslinking in all follicular lymphoma B cells, including all BTK -mutant follicular lymphoma. The augmented AKT phosphorylation following BCR crosslinking could be abrogated by pretreatment with a PI3Kδ inhibitor., Conclusions: Altogether, our data uncover novel unexpected properties of follicular lymphoma-associated BTK mutations with direct implications for targeted therapy development in follicular lymphoma. See related commentary by Afaghani and Taylor, p. 2123 ., (©2021 American Association for Cancer Research.)

Successful treatment of relapsed/refractory and rare subtypes of lymphomas remains a therapeutic challenge. Though the use of tumor profiling is increasing, little is described about how providers ultimately utilize this information in clinical decision-making. We reviewed 92 adult lymphoma patients who underwent an IRB-approved tumor sequencing protocol at the University of Michigan, MI-ONCOSEQ. Of this cohort, 60 had a targeted treatment suggested by their test results, and 11 patients ultimately underwent the MI-ONCOSEQ recommended therapy. One obtained complete response based on precision treatment recommendations. The two main barriers for targeted treatment utilization included inopportune timing (the patient either was sequenced too early or too late in their disease course) and clinical trial availability. While this study demonstrates the success of sequencing lymphomas for the identification of novel therapies, it also underlines the clinical challenges, namely the optimal timing and availability of trials, inherent in the successful application of this technology.

In relapsed/refractory Hodgkin lymphoma (R/R HL), immunotherapies such as the anti-programmed death-1 inhibitor pembrolizumab have demonstrated efficacy as monotherapy and are playing an increasingly prominent role in treatment. The CD30/CD16A-bispecific antibody AFM13 is an innate immune cell engager, a first-in-class, tetravalent antibody, designed to create a bridge between CD30 on HL cells and the CD16A receptor on natural killer cells and macrophages, to induce tumor cell killing. Early studies of AFM13 have demonstrated signs of efficacy as monotherapy for patients with R/R HL and the combination of AFM13 with pembrolizumab represents a rational new treatment modality. Here, we describe a phase 1b, dose-escalation study to assess the safety and preliminary efficacy of AFM13 in combination with pembrolizumab in patients with R/R HL. The primary objective was estimating the maximum tolerated dose; the secondary objectives were to assess safety, tolerability, antitumor efficacy, pharmacokinetics, and pharmacodynamics. In this heavily pretreated patient population, treatment with the combination of AFM13 and pembrolizumab was generally well tolerated, with similar safety profiles compared to the known profiles of each agent alone. The combination of AFM13 with pembrolizumab demonstrated an objective response rate of 88% at the highest treatment dose, with an 83% overall response rate for the overall population. Pharmacokinetic assessment of AFM13 in the combination setting revealed a half-life of up to 20.6 hours. This proof-of-concept study holds promise as a novel immunotherapy combination worthy of further investigation. This phase 1b study was registered at www.clinicaltrials.gov as NCT02665650., (© 2020 by The American Society of Hematology.)

Tenalisib (RP6530), a dual phosphoinositide 3-kinase δ/γ inhibitor was evaluated in a phase I/Ib study for maximum tolerated dose (MTD), pharmacokinetics, and efficacy in patients with relapsed/refractory peripheral and cutaneous T-Cell Lymphoma (TCL). Histologically confirmed (TCL) patients, with ≥1 prior therapy received Tenalisib orally in a 28-day cycle in doses of 200 to 800 mg twice daily (800 mg in fasting and fed state) in escalation phase ( n = 19) and 800 mg twice daily (fasting) in expansion phase ( n = 39). The most frequently reported treatment emergent adverse events (TEAE) and related TEAE were fatigue (45%) and transaminase elevations (33%), respectively. Most frequently reported related Grade ≥3 TEAE was transaminase elevation (21%). Two dose-limiting toxicities occurred in the 800 mg fed cohort; hence, 800 mg fasting dose was deemed MTD. Tenalisib was absorbed rapidly with a median half-life of 2.28 h. Overall response rate in 35 evaluable patients was 45.7% (3 complete response (CR); 13 partial response (PR)) and median duration of response was 4.9 months. Responding tumors showed a marked downregulation of CD30, IL-31 and IL-32α. With an acceptable safety and promising clinical activity, Tenalisib can be a potential therapeutic option for relapsed/refractory TCL. Currently, a phase I/II combination study with romidepsin is ongoing.

The CALGB/Alliance 50303 trial found a fivefold increase in the rate of severe neuropathies with DA-EPOCH-R compared to R-CHOP. A likely cause is a higher, uncapped dose of vincristine which is unique to DA-EPOCH-R. Due to a potential for increased toxicity and a paucity of literature confirming improved efficacy with higher vincristine doses, our institution implemented a 2 mg vincristine dose cap with DA-EPOCH-R. We conducted a single-center, retrospective cohort study assessing rates of neuropathy in patients receiving DA-EPOCH-R with or without a 2 mg vincristine dose cap. Patients who received a 2 mg vincristine dose cap had a significant reduction in the incidence of grade 2+ neuropathy (40.9% vs. 84.1%, p  = .001) and a significantly longer time to onset of grade 2+ neuropathy (not reached vs. 63 days, p  = .001). A vincristine dose cap of 2 mg per cycle may reduce the neurotoxicity-associated morbidity of DA-EPOCH-R.

Advanced classical Hodgkin lymphoma (cHL) is a rare lymphoid disease characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells. Each year, cHL accounts for 0.5% of all new cancer diagnoses and about 80% are diagnosed with advanced stage disease. Given the significant improvement in cure rates, the focus of treatment has shifted towards minimization of acute and long-term toxicities. PET-adapted strategies have largely been adopted as standard of care in the United States in an attempt to balance toxicities with adequate lymphoma control. However, the appropriate upfront chemotherapy regimen (ABVD versus eBEACOPP) remains controversial., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: There is an unmet need for antithrombotic treatments for venous thromboembolic disease that do not increase bleeding risk. Selectins are cell adhesion molecules that augment thrombosis by activating immune cells to initiate the coagulation cascade. GMI-1271, a potent small-molecule E-selectin antagonist, has been shown in mouse models to decrease thrombus burden with a low risk of bleeding., Methods: A first-in-human study of GMI-1271 was conducted to assess its safety, tolerability, and pharmacokinetic (PK) profile. As a secondary end point, biomarkers of coagulation, cell adhesion, and leukocyte/platelet activation were evaluated. Aims 1 and 2 were performed in healthy volunteers and evaluated single and multiple doses of the study drug, respectively. Aim 3 included 2 patients with isolated calf-level deep vein thrombosis (DVT)., Results: GMI-1271 showed consistent PK parameters for doses ranging from 2 to 40 mg/kg. Plasma levels increased in a linear manner with respect to dose, while clearance, volume of distribution, and half-life were not dose dependent. No accumulation was seen with multiple consecutive doses. No serious adverse events (grade 3 or 4) were reported. Biomarker analysis demonstrated a trend in reduction of soluble E-selectin (sEsel) levels with GMI-1271 exposure, while exposure did not impact laboratory testing of coagulation. Two patients with calf vein DVT were treated with GMI-1271 and demonstrated rapid improvement of symptoms after 48 hours, with repeat ultrasound showing signs of clot resolution., Conclusions: We demonstrate that GMI-1271 is safe in healthy volunteers and provide proof of concept that an E-selectin antagonist is a potential therapeutic approach to treat venous thrombosis., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals, Inc on behalf of International Society on Thrombosis and Haemostasis.)

Background: Hemophagocytic lymphohistiocytosis is a cytokine-driven inflammatory syndrome that is associated with substantial morbidity and mortality. Overall survival in adult patients with secondary haemophagocytic lymphohistiocytosis remains suboptimal, and novel therapeutic strategies are needed. The phosphorylation-dependent activation of the Janus family kinases JAK1 and JAK2 are hallmarks of the final common pathway in this disease. We therefore aimed to determine the activity and safety of ruxolitinib, a JAK inhibitor, in adults with secondary haemophagocytic lymphohistiocytosis., Methods: We performed an open-label, single-centre, pilot study of ruxolitinib in adults with secondary haemophagocytic lymphohistiocytosis at the University of Michigan Rogel Cancer Center (Ann Arbor, MI, USA). We included patients aged 18 years or more who fulfilled at least five of the eight HLH-2004 criteria for hemophagocytic lymphohistiocytosis. Discontinuation of corticosteroids was not required for enrolment in this study. Patients received oral ruxolitinib (15 mg twice a day) on a continuous 28-day cycle, or until disease progression or unacceptable toxicity. The primary endpoint was overall survival at 2 months from the first dose of ruxolitinib. Secondary endpoints included the assessment of adverse events, response (defined as the assessment of all quantifiable signs and laboratory abnormalities included in the diagnostic criteria for haemophagocytic lymphohistiocytosis), and pharmacodynamic biomarkers. Analyses were done in all treated patients with available data. This study is registered with ClinicalTrials.gov, number NCT02400463, and is still recruiting., Findings: As of Feb 7, 2019, five patients had been enrolled. The first patient was enrolled in February, 2016. No deaths were recorded, with a median follow-up of 490 days (IQR 190-1075). 2-month overall survival was 100% (95% CI 57-100). Regarding response, resolution of symptoms (either partial or complete) and disease-associated laboratory abnormalities was observed in all five patients. Cytopenias improved in all patients within the first week of treatment, leading to relatively rapid transfusion independence, discontinuation of corticosteroids, and hospital discharge. A single serious adverse event (ie, grade 4 febrile neutropenia) was reported. One patient discontinued treatment because of grade 2 extremity pain and no treatment-related deaths were observed. Improvements in inflammatory markers (eg, ferritin, soluble IL-2 receptor) and T cells and monocytes activation (ie, decreased STAT1 phosphorylation) were observed following treatment., Interpretation: These preliminary data suggest that ruxolitinib is active, well tolerated, and manageable in the outpatient setting in patients with secondary haemophagocytic lymphohistiocytosis. Given the paucity of effective, non-myelosuppressive therapies, these preliminary findings have important therapeutic implications for patients with haemophagocytic lymphohistiocytosis and other cytokine-release syndromes and warrant further investigation., Funding: National Cancer Institute, the University of Michigan Rogel Cancer Center, and Incyte Corporation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intravascular large B-cell lymphoma (IVLBCL) is a rare entity, with a generally aggressive course that may vary based on geographic presentation. While a United States (US) registry study showed relatively good outcomes with IVLBCL, clinicopathological and treatment data were unavailable. We performed a detailed retrospective review of cases identified at 8 US medical centres, to improve understanding of IVLBCL and inform management. We compiled data retrieved via an Institutional Review Board-approved review of IVLBCL cases identified from 1999 to 2015 at nine academic institutions across the US. We characterized the cohort's clinical status at time of diagnosis, presenting diagnostic and clinical features of the disease, treatment modalities used and overall prognostic data. Our cohort consisted of 54 patients with varying degrees of clinical features. Adjusting for age, better performance status at presentation was associated with increased survival time for the patients diagnosed in vivo (hazard ratio: 2·12, 95% confidence interval 1·28, 3·53). Based on the data we have collected, it would appear that the time interval to diagnosis is a significant contributor to outcomes of patients with IVLBCL., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

Optimal salvage therapy for primary refractory peripheral T-cell lymphomas (PTCL) and the role of hematopoietic stem cell transplant (SCT) remain poorly defined. We conducted a retrospective review of clinical outcomes and prognostic factors in a single-center cohort of 93 patients with primary refractory PTCL, defined as progression during first-line therapy or relapse within 6 months of its completion. Clinical outcomes were poor in this population, with median event-free survival (EFS) of 3.5 months, median overall survival (OS) of 9.1 months, and 34% 3-year survival. Outcomes were comparable in patients who progressed through first-line therapy and patients who achieved CR/PR and subsequently relapsed within 6 months. A majority exhibited high-risk features and had intermediate to high risk IPI, which correlated with inferior outcomes. There was no difference in outcomes between patients who received single-agent salvage regimens and patients who underwent traditional, multi-agent salvage regimens. Thus, participation in well-designed clinical trials should be encouraged in this population. Additionally, there may be a trend toward improved EFS and OS in patients who underwent autologous or allogeneic SCT compared to patients who achieved CR or PR but were not transplanted., (© 2017 Wiley Periodicals, Inc.)

Survival following anthracycline-based chemotherapy remains poor among patients with most T-cell lymphoproliferative disorders. This may be attributed, at least in part, to cell-autonomous mechanisms of chemotherapy resistance observed in these lymphomas, including the loss of important tumor suppressors and the activation of signaling cascades that culminate in the expression and activation of transcription factors promoting cell growth and survival. Therefore, the identification of novel therapeutic targets is needed. In an effort to identify novel tumor dependencies, we performed a loss-of-function screen targeting ≈500 kinases and identified polo-like kinase 1 (PLK-1). This kinase has been implicated in the molecular cross-talk with important oncogenes, including c-Myc, which is itself an attractive therapeutic target in subsets of T-cell lymphomas and in high-grade ("double hit") diffuse large B-cell lymphomas. We demonstrate that PLK-1 expression is prevalent among these aggressive lymphomas and associated with c-myc expression. Importantly, PLK-1 inhibtion with the PLK-1 inhibitor volasertib significantly reduced downstream c-myc phosphorylation and impaired BRD4 binding to the c-myc gene, thus inhibiting c-myc transcription. Therefore, volasertib led to a nearly complete loss of c-myc expression in cell lines and tumor xenografts, induced apoptosis, and thus warrants further investigation in these aggressive lymphomas., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Influenza morbidity and mortality can be severe and costly. Vaccination rates remain suboptimal in cancer patients due to provider- and patient-related factors. The objective of this study was to evaluate whether low-cost provider- and patient-focused interventions would increase influenza vaccination rates at the University of Michigan Comprehensive Cancer Center (UMCCC). This quality improvement project included all patients without documentation of influenza vaccination prior to their first outpatient appointment during the 2011-2012 and 2012-2013 influenza seasons. The multi-stepped intervention included provider and patient reminders. Influenza vaccination rates were compiled using CPT-4 codes. Same-day (with appointment) vaccination rates during the intervention seasons were compared to historical (2005-2011 seasons) controls; vaccination rates were also compared to contemporary control population at the University of Michigan Health System (UMHS). Reasons for non-adherence with vaccination were explored. The cumulative same-day vaccination rate in eligible adults was 10.1 % (2011-2012) and 9.4 % (2012-2013) compared to an average 6.9 % during influenza seasons 2005-2011. Based on logistic regression analysis, there was a 37.6 % (95 % CI 35-40.3 %) and 56.1 % (95 % CI 40.9-73 %) relative increase in the adult vaccination rate associated with the intervention, with 399 and 697 additional vaccinations, respectively, for each season. During the 2012-2013 season, the UMCCC adult vaccination rate was higher compared to the remainder of that of the UMHS. The intervention was well accepted by providers. Reasons for no vaccination were provider- and patient-related. Increasing provider and patient awareness with a simple, inexpensive intervention was associated with higher influenza vaccination rates at a large academic cancer center. The intervention is permanently implemented during influenza seasons.

The transcription factor GATA-3, highly expressed in many cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphomas (PTCL), confers resistance to chemotherapy in a cell-autonomous manner. As GATA-3 is transcriptionally regulated by NF-κB, we sought to determine the extent to which proteasomal inhibition impairs NF-κB activation and GATA-3 expression and cell viability in malignant T cells. Proteasome inhibition, NF-κB activity, GATA-3 expression, and cell viability were examined in patient-derived cell lines and primary T-cell lymphoma specimens ex vivo treated with the oral proteasome inhibitor ixazomib. Significant reductions in cell viability, NF-κB activation, and GATA-3 expression were observed preclinically in ixazomib-treated cells. Therefore, an investigator-initiated, single-center, phase II study with this agent in patients with relapsed/refractory CTCL/PTCL was conducted. Concordant with our preclinical observations, a significant reduction in NF-κB activation and GATA-3 expression was observed in an exceptional responder following one month of treatment with ixazomib. While ixazomib had limited activity in this small and heterogeneous cohort of patients, inhibition of the NF-κB/GATA-3 axis in a single exceptional responder suggests that ixazomib may have utility in appropriately selected patients or in combination with other agents., (© 2017 Wiley Periodicals, Inc.)

The T-cell lymphoproliferative disorders are a heterogeneous group of non-Hodgkin's lymphomas (NHL) for which current therapeutic strategies are inadequate, as most patients afflicted with these NHL will succumb to disease progression within 2 years of diagnosis. Appreciation of the genetic and immunologic landscape of these aggressive NHL, including PD-L1 (B7-H1, CD274) expression by malignant T cells and within the tumor microenvironment, provides a strong rationale for therapeutic targeting this immune checkpoint. While further studies are needed, the available data suggests that responses with PD-1 checkpoint blockade alone will unlikely approach those achieved in other lymphoproliferative disorders. Herein, we review the unique challenges posed by the T-cell lymphoproliferative disorders and discuss potential strategies to optimize checkpoint blockade in these T-cell derived malignancies.

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of extranodal lymphomas involving the skin. Diagnosis of the two main subtypes of CTCL-mycosis fungoides (MF) and Sézary syndrome (SS)-is based on the International Society for Cutaneous Lymphomas/European Organization for Research and Treatment of Cancer (ISCL/EORTC) classification system, which utilizes clinical, histopathological, molecular biologic, and immunopathologic features. Risk stratification, based on TNMB (tumor, node, metastasis, and blood) staging, provides prognostic information, with limited-stage disease conferring the longest median overall survival. Skin-directed therapies are preferred in the management of limited-stage disease, whereas advanced-stage disease requires systemic therapies. As the mechanisms of CTCL pathogenesis are increasingly understood, new monoclonal antibodies, checkpoint inhibitors, immunomodulatory agents, and small molecules are under investigation and may provide additional therapeutic options for those with advanced CTCL. This review examines the current landscape of targeted therapies in the treatment of CTCLs.

Desmoid tumors are rare, clonal collections of benign fibrous tissue that exhibit a highly variable clinical course. This article presents a comprehensive review of desmoid tumors and summarizes the current literature pertaining to clinical presentation, diagnostic modalities, pathogenesis, prognostic factors, and management options., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Fine-needle aspiration biopsy (FNA) of the abdominal fat pad is a minimally invasive procedure to demonstrate tissue deposits of amyloid. However, protocols to evaluate amyloid in fat pad aspirates are not standardized, especially for detecting scant amyloid in early disease., Materials and Methods: We studied abdominal fat pad aspirates from 33 randomly selected patients in whom subsequent tissue biopsy, autopsy, and/or medical history for confirmation of amyloidosis (AL) were also available. All these cases were suspected to have early AL, but had negative results on abdominal fat pad aspirates evaluated by polarizing microscopy of Congo Red stained sections (CRPM). The results with CRPM between four reviewers were compared in 12 cases for studying inter observer reproducibility. 24 cases were also evaluated by ultrastructural study with electron microscopy (EM)., Results: Nine of thirty-three (27%) cases reported negative by polarizing microscopy had amyloidosis. Reanalysis of 12 mixed positive-negative cases, showed considerable inter-observer variability with frequent lack of agreement between four observers by CRPM alone (Cohen's Kappa index of 0.1, 95% CI -0.1 to 0.36). EM showed amyloid in the walls of small blood vessels in fibroadipose tissue in four out of nine cases (44%) with amyloidosis., Conclusion: In addition to poor inter-observer reproducibility, CRPM alone in cases with scant amyloid led to frequent false negative results (9 out of 9, 100%). For improved detection of AL, routine ultrastructural evaluation with EM of fat pad aspirates by evaluating at least 15 small blood vessels in the aspirated fibroadipose tissue is recommended. Given the high false negative rate for CRPM alone in early disease, routine reflex evaluation with EM is highly recommended to avert the invasive option of biopsying various organs in cases with high clinical suspicion for AL.

A 60-year-old Caucasian female presented with stage IIA (T2N0M0) estrogen- and progesterone-negative and HER2-positive breast cancer. She was started on an adjuvant chemotherapy regimen of docetaxel, carboplatin, and trastuzumab (TCH). She tolerated the first two cycles of the TCH regimen well. However, 3-4 days after the third and fourth cycles, she developed acute pancreatitis. Elevated pancreatic enzymes and abdominal computed tomography (CT) imaging findings confirmed the diagnosis of acute pancreatitis. Common causes of pancreatitis were ruled out. Given the time course it was assumed that the chemotherapy was the cause of pancreatitis in our patient. The patient did not receive any further docetaxel and carboplatin chemotherapy but continued on adjuvant trastuzumab alone for a planned duration of 1 year without any recurrence of acute pancreatitis.

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Members of the transforming growth factor-beta superfamily, including bone morphogenetic protein 4 (BMP-4), have been implicated as regulators of neuronal and glial differentiation. To test for a possible role of BMP-4 in early mammalian neural specification, we examined its effect on neurogenesis in aggregate cultures of mouse embryonic stem (ES) cells. Compared to control aggregates, in which up to 20% of the cells acquired immunoreactivity for the neuron-specific antibody TuJ1, aggregates maintained for 8 days in serum-free medium containing BMP-4 generated 5- to 10-fold fewer neurons. The action of BMP-4 was dose dependent and restricted to the fifth through eighth day in suspension. In addition to the reduction in neurons, we observed that ES cell cultures exposed to BMP-4 contained fewer cells that were immunoreactive for glial fibrillary acidic protein or the HNK-1 neural antigen. Furthermore, under phase contrast, cultures prepared from BMP-4-treated aggregates contained a significant proportion of nonneuronal cells with a characteristic flat, elongated morphology. These cells were immunoreactive for antibodies to the intermediate filament protein vimentin; they were rare or absent in control cultures. Treatment with BMP-4 enhanced the expression of the early mesodermal genes brachyury and tbx6 but had relatively little effect on total cell number or cell death. Coapplication of the BMP-4 antagonist noggin counteracted the effect of exogenous BMP-4, but noggin alone had no effect on neuralization in either the absence or presence of retinoids. Collectively, our results suggest that BMP-4 can overcome the neuralizing action of retinoic acid to enhance mesodermal differentiation of murine ES cells., (Copyright 1999 John Wiley & Sons, Inc.)

Metastatic Lewis lung carcinoma (LLC-LN7) cells have increased protein kinase A (PKA) activity and are more invasive in vitro than are non-metastatic (LLC-C8) cells. To determine whether PKA mediates the in vitro invasiveness and in vivo metastatic capabilities of these tumor cells, the LLC variants were stably transfected to over-express the C alpha subunit of PKA, and thus to have increased PKA activity, or to express a mutant cAMP-resistant PKA R1 alpha subunit which blocks PKA activation. Wild-type LLC-LN7 tumor cells were invasive in vitro and in vivo, recurred after tumor excision and metastasized to the lungs. However, they lost these properties after transfection to express the mutant R1 alpha that blocks PKA activation. The non-invasive, non-recurring and non-metastatic LLC-C8 cells gained the capacity to invade, to recur following tumor excision and to metastasize when transfected to express the PKA C alpha subunit.

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) by metastatic Lewis lung carcinoma cells (LLC-LN7) was previously shown to contribute to the maintenance of phenotypic characteristics associated with an increased capacity to metastasize. In the present study, pre-incubation of LLC-LN7 cells with neutralizing anti-GM-CSF antibodies diminished the capacity of the tumor cells to form experimental metastases after i.v. inoculation, while pre-incubation with recombinant GM-CSF (rGM-CSF) increased formation of metastases. In the presence of rGM-CSF, the LLC-LN7 cells exhibited an increased capacity to migrate, invade through a reconstituted basement membrane, and adhere to lung tissue. Studies to identify the signal transduction pathway through which GM-CSF enhanced the in vitro metastatic properties of the LLC-LN7 tumor cells implicated protein kinase A (PKA). Signaling through PKA was suggested by the demonstration that the stimulation of tumor-cell motility by GM-CSF was blocked in the presence of the adenylate cyclase inhibitor nicotinic acid, or the PKA inhibitors A3 or KT5720. In addition, the role of PKA as a signaling mechanism for GM-CSF was assessed by using REV-LN7 cells, which are LLC-LN7 cells that have been stably transfected with an expression vector encoding a mutant PKA RI alpha subunit and which, in turn, express a cAMP-resistant PKA. Adherence and invasion by the PKA-defective REV-LN7 cells were not stimulated by rGM-CSF, contrasting with the stimulation observed for wild-type LLC-LN7 cells. These data suggest that rGM-CSF can further enhance the in vitro metastatic characteristics of LLC-LN7 tumor cells and that this is dependent on signal transduction through PKA.

Production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by metastatic Lewis lung carcinoma tumors (LLC-LN7) has been shown to increase their migration and invasion in vitro, and metastasis in vivo. The present studies showed that treatment of the LLC-LN7 cells by 2 days culture with 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited their production of GM-CSF. The 1,25(OH)2D3-treated cells also became less migratory and invasive. This inhibitory effect on tumor motility could not be readily reversed upon removal of 1,25(OH)2D3. The mechanism by which 1,25(OH)2D3 reduced tumor motility was attributed to the inhibition of tumor GM-CSF production since the capacity to migrate and invade could be restored by supplementing the medium with recombinant GM-CSF. In vivo studies showed that treatment of LLC-LN7-bearing mice with 1,25(OH)2D3 had no effect on growth of s.c. primary tumors, but reduced the formation of tumor metastases. These results show that 1,25(OH)2D3 can interrupt the autocrine motility-stimulation cascade by reducing tumor production of GM-CSF, thus reducing the expression of properties that are characteristic of metastatic tumor cells.