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We describe significant improvements to a well-established method of obtaining optical sectioning in a conventional wide-field microscope. This method relies on the projection of a single-frequency grid pattern onto an object followed by mathematical manipulation of three images taken with the grid in different phases. Here, we present the use of Bayesian Spectral Analysis to determine accurate estimates of the phase of the grid pattern, permitting rapid and precise calibration of grid location. In common with previous algorithms, multiple images are combined to produce both an optical section and a wide-field image. We describe innovations such as the use of non-uniform phase or least-squares solutions involving more than three images, in conjunction with direct phase estimates obtained using Bayesian Spectral Analysis, to yield images substantially free of artefacts. Auxiliary results, such as a method for determining the tilt of the grid, are also presented. [ABSTRACT FROM AUTHOR]

Background: Sarcomatoid renal cell carcinoma (RCC) is associated with an aggressive biology and a poor prognosis. Poor-risk RCC is defined by clinical prognostic factors and demonstrates similarly aggressive behavior. No standard treatment exists for patients with sarcomatoid RCC, and treatment options for patients with poor-risk disease are of limited benefit. The objective of this study was to investigate the efficacy of antiangiogenic therapy in combination with cytotoxic chemotherapy in clinically aggressive RCC.Methods: This was a phase 2, single-arm trial of sunitinib and gemcitabine in patients with sarcomatoid or poor-risk RCC. The primary end point was the objective response rate (ORR). Secondary end points included the time to progression (TTP), overall survival (OS), safety, and biomarker correlatives.Results: Overall, 39 patients had sarcomatoid RCC, and 33 had poor-risk RCC. The ORR was 26% for patients with sarcomatoid RCC and 24% for patients with poor-risk RCC. The median TTP and OS for patients with sarcomatoid RCC were 5 and 10 months, respectively. For patients with poor-risk disease, the median TTP and OS were 5.5 and 15 months, respectively. Patients whose tumors had>10% sarcomatoid histology had a higher clinical benefit rate (ORR plus stable disease) than those with≤10% sarcomatoid histology (P = 0.04). The most common grade 3 or higher treatment-related adverse events included neutropenia (n = 20), anemia (n = 10), and fatigue (n = 7).Conclusions: These results suggest that antiangiogenic therapy and cytotoxic chemotherapy are an active and well-tolerated combination for patients with aggressive RCC. The combination may be more efficacious than either therapy alone and is currently under further investigation. [ABSTRACT FROM AUTHOR]

Prostate cancer represents a major global health challenge, necessitating efficacious therapeutic strategies. Androgen receptor pathway inhibitors (ARPIs) have become central to prostate cancer treatment, demonstrating significant effectiveness in both metastatic and non-metastatic contexts. Abiraterone acetate, by inhibiting androgen synthesis, deprives cancer cells androgens necessary for growth, while second-generation androgen receptor (AR) antagonists disrupt AR signaling by blocking AR binding, thereby impeding tumor progression. Given the predominance of prostate cancer in the elderly, who often present with multiple comorbidities requiring complex pharmacological regimens, the potential for drug-drug interactions with ARPIs is a critical concern. These interactions, particularly through pathways like CYP2D6 inhibition by abiraterone and CYP3A4 induction by enzalutamide and apalutamide, necessitate a thorough understanding to optimize therapeutic outcomes and minimize adverse effects. This review aims to delineate the efficacy of ARPIs in prostate cancer management and elucidate their interaction with common medications, highlighting the importance of vigilant drug management to optimize patient care., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Lymphocytes are closely linked to mechanisms of action of immuno-oncology (IO) agents. We aimed to assess the prognostic significance of absolute lymphocyte count (ALC) in patients with metastatic renal cell carcinoma (mRCC)., Patients and Methods: Using the International mRCC Database Consortium (IMDC), patients receiving first-line IO-based combination therapy were analysed. Baseline patient characteristics, objective response rates (ORRs), time to next treatment (TTNT), and overall survival (OS) were compared., Results: Of 966 patients included, 195 (20%) had lymphopenia at baseline, and they had a lower ORR (37% versus 45%; P < 0.001), shorter TTNT (10.1 months versus 24.3 months; P < 0.001), and shorter OS (30.4 months versus 48.2 months; P < 0.001). Among 125 patients with lymphopenia at baseline, 52 (42%) experienced ALC recovery at 3 months, and they had longer OS (not reached versus 30.4 months; P = 0.012). On multivariable analysis for OS, lymphopenia was an independent adverse prognostic factor (hazard ratio 1.68; P < 0.001). Incorporation of lymphopenia into the IMDC criteria improved OS prediction accuracy (C-index from 0.688 to 0.707)., Conclusions: Lymphopenia was observed in one-fifth of treatment-naive patients with mRCC and may serve as an indicator of unfavourable oncologic outcomes in the contemporary IO era., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The isolation of cell-free DNA (cfDNA) from the bloodstream can be used to detect and analyze somatic alterations in circulating tumor DNA (ctDNA), and multiple cfDNA-targeted sequencing panels are now commercially available for Food and Drug Administration (FDA)-approved biomarker indications to guide treatment. More recently, cfDNA fragmentation patterns have emerged as a tool to infer epigenomic and transcriptomic information. However, most of these analyses used whole-genome sequencing, which is insufficient to identify FDA-approved biomarker indications in a cost-effective manner., Patients and Methods: We used machine learning models of fragmentation patterns at the first coding exon in standard targeted cancer gene cfDNA sequencing panels to distinguish between cancer and non-cancer patients, as well as the specific tumor type and subtype. We assessed this approach in two independent cohorts: a published cohort from GRAIL (breast, lung, and prostate cancers, non-cancer, n = 198) and an institutional cohort from the University of Wisconsin (UW; breast, lung, prostate, bladder cancers, n = 320). Each cohort was split 70%/30% into training and validation sets., Results: In the UW cohort, training cross-validated accuracy was 82.1%, and accuracy in the independent validation cohort was 86.6% despite a median ctDNA fraction of only 0.06. In the GRAIL cohort, to assess how this approach performs in very low ctDNA fractions, training and independent validation were split based on ctDNA fraction. Training cross-validated accuracy was 80.6%, and accuracy in the independent validation cohort was 76.3%. In the validation cohort where the ctDNA fractions were all <0.05 and as low as 0.0003, the cancer versus non-cancer area under the curve was 0.99., Conclusions: To our knowledge, this is the first study to demonstrate that sequencing from targeted cfDNA panels can be utilized to analyze fragmentation patterns to classify cancer types, dramatically expanding the potential capabilities of existing clinically used panels at minimal additional cost., Competing Interests: Disclosure KTH has a family member who is an employee of Epic Systems. MLB has a family member who is an employee of Luminex. SGZ reports unrelated patents licensed to Veracyte, and that a family member is an employee of Artera and holds stock in Exact Sciences. KTH, SGZ, and the University of Wisconsin have filed a provisional patent on the work herein. SMD reports consulting relationships with BMS, Oncternal therapeutics, Janssen R&D/J&J and a grant from Pfizer/Astellas/Medivation (the grant was submitted to Medivation, ultimately funded by Astellas and then moved to Pfizer). FYF reports personal fees from Janssen Oncology, Bayer, PFS Genomics, Myovant Sciences, Roivant Sciences, Astellas Pharma, Foundation Medicine, Varian, Bristol Myers Squibb (BMS), Exact Sciences, BlueStar Genomics, Novartis, and Tempus; other support from Serimmune and Artera outside the submitted work. Integrated DNA Technologies (IDT, Coralville, IA) assisted in a pilot project to assess the performance characteristics of the UW panel before purchase, but played no role in this study. All other authors have declared no conflicts of interest. Data Sharing Raw sequencing data from the GRAIL dataset are available at the European Genome Archive (Dataset ID EGAD00001005302). Our institutional protocol does not allow unrestricted public access to the raw sequencing data. Therefore, data sharing requests must be submitted to the University of Wisconsin-Madison for approval. For samples from the two clinical trials (NCT03090165, NCT03725761), these trials are still ongoing, and data sharing requests must be submitted to the trial organizers., (Published by Elsevier Ltd.)

Background: Vaccination is an important preventive health measure to protect against symptomatic and severe COVID-19. Impaired immunity secondary to an underlying malignancy or recent receipt of antineoplastic systemic therapies can result in less robust antibody titers following vaccination and possible risk of breakthrough infection. As clinical trials evaluating COVID-19 vaccines largely excluded patients with a history of cancer and those on active immunosuppression (including chemotherapy), limited evidence is available to inform the clinical efficacy of COVID-19 vaccination across the spectrum of patients with cancer., Patients and Methods: We describe the clinical features of patients with cancer who developed symptomatic COVID-19 following vaccination and compare weighted outcomes with those of contemporary unvaccinated patients, after adjustment for confounders, using data from the multi-institutional COVID-19 and Cancer Consortium (CCC19)., Results: Patients with cancer who develop COVID-19 following vaccination have substantial comorbidities and can present with severe and even lethal infection. Patients harboring hematologic malignancies are over-represented among vaccinated patients with cancer who develop symptomatic COVID-19., Conclusions: Vaccination against COVID-19 remains an essential strategy in protecting vulnerable populations, including patients with cancer. Patients with cancer who develop breakthrough infection despite full vaccination, however, remain at risk of severe outcomes. A multilayered public health mitigation approach that includes vaccination of close contacts, boosters, social distancing, and mask-wearing should be continued for the foreseeable future., Competing Interests: Disclosure ALS reports non-financial support from Astellas, non-financial support from Pfizer, outside the submitted work. CL reports research support from Genentech/imCORE, outside the submitted work. ZB reports non-financial support from Bristol Myers Squibb, grants from Genentech/imCORE, personal fees from UpToDate, outside the submitted work. CYH reports personal fees from NashBio, outside the submitted work. SAB reports personal fees from Exelixis, personal fees from Seattle Genetics, personal fees from Pfizer, personal fees from Bristol Myers Squibb, outside the submitted work. CRF reports research grants from the Merck Foundation and National Comprehensive Cancer Network (NCCN)/Pfizer, outside the submitted work. EAG reports personal fees and other from Alexion Pharmaceuticals, personal fees and non-financial support from Novartis Pharmaceuticals, personal fees, non-financial support and other from Astex/Otsuka Pharmaceuticals, other from Apellis Pharmaceuticals, personal fees, non-financial support, and other from Celgene/Bristol Myers Squibb, grants and personal fees from AbbVie/Genentech, other from Celldex Therapeutics, personal fees from Boston Biomedical, outside the submitted work. JEH reports research funding paid to her institution from Dendreon Pharmaceuticals LLC, research funding paid to her institution from Regeneron Pharmaceuticals, personal fees from Genzyme, personal fees from Seagen, outside the submitted work. RRM reports grants and personal fees from Bayer, grants from Pfizer, grants from Tempus, personal fees from AVEO, personal fees from Caris, personal fees from Bristol Myers Squib, personal fees from Exelixis, personal fees from Janssen, personal fees from Novartis, personal fees from Pfizer, personal fees from Sanofi, personal fees from Tempus, personal fees from Dendreon, personal fees from Vividion, personal fees from AstraZeneca, personal fees from Calithera, personal fees from Merck, outside the submitted work. OAP reports personal fees from International Consulting Associates, Inc., outside the submitted work. EMW-B reports personal fees from Astellas, personal fees from AVEO Oncology, personal fees from Bristol Myers Squibb, other from Exelixis, grants from Pfizer Global Medical Grants, other from Nektar, other from Immunomedics, outside the submitted work. SM reports personal fees from National Geographic, outside the submitted work. DF reports a grant from Merck to study COVID-19 in immunocompromised patients, outside of the submitted work. YS reports personal fees from Novartis, personal fees from Roche, personal fees from Pfizer, personal fees from Janssen, personal fees from Eisai, personal fees from AstraZeneca, outside of the submitted work. JLW reports personal fees from Westat, personal fees from Roche, personal fees from Melax Tech, personal fees from Flatiron Health, other from HemOnc.org LLC (ownership), outside the submitted work. TKC reports institutional and personal, paid and unpaid support for research, advisory boards, consultancy, and honoraria from AstraZeneca, Aravive, Aveo, Bayer, Bristol Myers Squibb, Eisai, EMD Serono, Exelixis, GlaxoSmithKline, IQVIA, Ipsen, Kanaph, Lilly, Merck, Nikang, Novartis, Pfizer, Roche, Sanofi/Aventis, Takeda, Tempest, UpToDate, CME events (Peerview, OncLive and others), outside the submitted work. All other authors have declared no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Patients with cancer may be at high risk of adverse outcomes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We analyzed a cohort of patients with cancer and coronavirus 2019 (COVID-19) reported to the COVID-19 and Cancer Consortium (CCC19) to identify prognostic clinical factors, including laboratory measurements and anticancer therapies., Patients and Methods: Patients with active or historical cancer and a laboratory-confirmed SARS-CoV-2 diagnosis recorded between 17 March and 18 November 2020 were included. The primary outcome was COVID-19 severity measured on an ordinal scale (uncomplicated, hospitalized, admitted to intensive care unit, mechanically ventilated, died within 30 days). Multivariable regression models included demographics, cancer status, anticancer therapy and timing, COVID-19-directed therapies, and laboratory measurements (among hospitalized patients)., Results: A total of 4966 patients were included (median age 66 years, 51% female, 50% non-Hispanic white); 2872 (58%) were hospitalized and 695 (14%) died; 61% had cancer that was present, diagnosed, or treated within the year prior to COVID-19 diagnosis. Older age, male sex, obesity, cardiovascular and pulmonary comorbidities, renal disease, diabetes mellitus, non-Hispanic black race, Hispanic ethnicity, worse Eastern Cooperative Oncology Group performance status, recent cytotoxic chemotherapy, and hematologic malignancy were associated with higher COVID-19 severity. Among hospitalized patients, low or high absolute lymphocyte count; high absolute neutrophil count; low platelet count; abnormal creatinine; troponin; lactate dehydrogenase; and C-reactive protein were associated with higher COVID-19 severity. Patients diagnosed early in the COVID-19 pandemic (January-April 2020) had worse outcomes than those diagnosed later. Specific anticancer therapies (e.g. R-CHOP, platinum combined with etoposide, and DNA methyltransferase inhibitors) were associated with high 30-day all-cause mortality., Conclusions: Clinical factors (e.g. older age, hematological malignancy, recent chemotherapy) and laboratory measurements were associated with poor outcomes among patients with cancer and COVID-19. Although further studies are needed, caution may be required in utilizing particular anticancer therapies., Clinical Trial Identifier: NCT04354701., Competing Interests: Disclosure JDA reports research funding to the institution from Tesaro, outside the submitted work. ZB reports nonfinancial support from Bristol Myers Squibb and grants from Genentech/imCORE, outside the submitted work. BB reports research funding to the institution from Boehringer Ingelheim, Bicycle Therapeutics, Syros Pharmaceuticals, and Ikena Oncology, all outside the submitted work. TB-S reports research funding to the institution from Agios, Arys, Boston Biomedical, Bayer, Amgen, Merck, Celgene, Lilly, Ipsen, Clovis, Seattle Genetics, Array Biopharma, Genentech, Novartis, Mirati, Merus, AbGenomics, Incyte, Pfizer, BMS; consulting (to institution) for Ipsen, Array Biopharma, Pfizer, Seattle Genetics, Bayer, Genentech, Incyte, and Merck; consulting (to self) for AbbVie, Boehringer Ingelheim, Janssen, Eisai, Daiichi Sankyo, Natera, Treos Bio, Celularity, Exact Science, Sobi, BeiGene, Xilis, Astra Zeneca, and Foundation Medicine; serving on Independent Data Monitoring Committee/Data and Safety Monitoring Board (to self) for AstraZeneca, Exelixis, Lilly, PanCAN, and 1Globe; positions on Scientific Advisory Board for Imugene, Immuneering, and Sun Biopharma; and inventions/patents (WO/2018/183488 and WO/2019/055687), all outside the submitted work. SB reports being on advisory boards for Bristol Meyers Squibb and Seattle Genetics. MAB reports personal fees from Exelixis, Bristol-Myers Squibb, Bayer, Eisai, Pfizer, AstraZeneca, Janssen, Genomic Health, Nektar, and Sanofi; grants from Xencor, Bayer, Bristol-Myers Squibb, Genentech/Roche, Seattle Genetics, Incyte, Nektar, AstraZeneca, Tricon Pharmaceuticals, Peloton Therapeutics, and Pfizer, outside the submitted work. NB reports honoraria from Novartis, Pfizer, Roche, and Lilly, outside the submitted work. DWB reports research funding to the institution from Exelixis, Ayala, Merck, and Elevar, all outside the submitted work. DDC declares consulting or advisory role with Exelixis, outside the submitted work. TKC reports institutional and personal research support from Alexion, Analysis Group, AstraZeneca, Aveo, Bayer, Bristol Myers-Squibb/ER Squibb and sons LLC, Calithera, Cerulean, Corvus, Eisai, Exelixis, F. Hoffmann-La Roche, Foundation Medicine Inc., Genentech, GlaxoSmithKline, Ipsen, Lilly, Merck, Novartis, Peloton, Pfizer, Prometheus Labs, Roche, Roche Products Limited, Sanofi/Aventis, Takeda, Tracon; consulting/honoraria or advisory role with Alexion, Analysis Group, AstraZeneca, Aveo, Bayer, Bristol Myers-Squibb/ER Squibb and sons LLC, Cerulean, Corvus, Eisai, EMD Serono, Exelixis, Foundation Medicine Inc., Genentech, GlaxoSmithKline, Heron Therapeutics, Infinity Pharma, Ipsen, Jansen Oncology, IQVIA, Lilly, Merck, NCCN, Novartis, Peloton, Pfizer, Pionyr, Prometheus Labs, Roche, Sanofi/Aventis, Surface Oncology, Tempest, Up-to-Date; CME-related events (e.g. OncLive, PVI, MJH Life Sciences); stock ownership in Pionyr, Tempest; patents filed, royalties, or other intellectual properties related to biomarkers of immune checkpoint blockers; fees for travel, accommodations, expenses, medical writing in relation to consulting, advisory roles, or honoraria; and no speaker's bureau; also supported in part by the Dana-Farber/Harvard Cancer Center Kidney SPORE and Program, the Kohlberg Chair at Harvard Medical School and the Trust Family, Michael Brigham, and Loker Pinard Funds for Kidney Cancer Research at DFCI. DBD reports consulting for Ipsen, Boehringer Ingelheim; ASCO Young Investigator Award from Conquer Cancer Foundation, outside the submitted work. AE reports grant support from AstraZeneca, outside the submitted work. DF reports research funding to the institution from Viracor-Eurofins and Astellas, all outside the submitted work. LAF reports clinical trial funding to the institution from BMS, EMD Serono, Pfizer, Merck KGaA, Array, Kartos, Merck, and Incyte, ECOG-ACRIN study funding from Array; and personal fees from Elsevier and Via Oncology, outside the submitted work. DBF reports honoraria from Castle Biosciences. SMG reports Honoraria from AstraZeneca, Merck, Genentech/Roche; consulting or advisory role with Genentech/Roche, AstraZeneca, Bristol-Myers Squibb, Takeda, Xcovery, Boehringer Ingelheim, Novocure, Daiichi Sankyo, Novartis, Jazz Pharmaceuticals, Blueprint Medicines, Eli Lilly, Pfizer, Janssen Oncology; research funding (to self) from Merck, AstraZeneca; research funding (to institution) from Genentech/Roche, Merck, Blueprint Medicines, ARIAD/Takeda, Astellas Pharma, Lycera, Daiichi Sankyo, IMAB, Nektar, AstraZeneca, Pfizer, Amgen; travel, accommodations, expenses from Genentech/Roche, Merck; and other relationship from AstraZeneca, all outside the submitted work. MDG reports personal fees from Genentech, Pfizer, Astra Zeneca, Merck, Bristol Myers Squib, Dragonfly, Dracen, Seattle Genetics, and Astellas, outside the submitted work. PG reports consulting fees from AstraZeneca, Bayer, Bristol-Myers Squibb, Clovis Oncology, Dyania Health, Driver, EMD Serono, Exelixis, Foundation Medicine, Genentech/Roche, Genzyme, GlaxoSmithKline, Heron Therapeutics, Immunomedics, Infinity Pharmaceuticals, Janssen, Merck, Mirati Therapeutics, Pfizer, Seattle Genetics, QED Therapeutics; research funding to institution from Merck, Mirati Therapeutics, Pfizer, Clovis Oncology, Bavarian Nordic, Immunomedics, Debiopharm, Bristol-Myers Squibb, QED Therapeutics, GlaxoSmithKline, and Kure It Cancer Research, all outside the submitted work. SG reports research funding to the institution from AstraZeneca and consulting/advisory role with Puma Biotechnology. SG reports consultancy fees from BMS, Merck, AstraZeneca, Seattle Genetics, Pfizer; and speaker fees from Seattle Genetics and Janssen, all outside the submitted work. TRH reports consulting or advisory role with Curium, ScioScientific, TERUMO, Lexicon, Ipsen, Advanced Accelerator; research funding from Ipsen, ArQule, Agios, Thermo Fisher Scientific, Basilea. BH reports research funding to the institution from Amgen, AbbVie, BI, Mirati, Merck, Eli-Lilly, AstraZeneca, BMS, Novartis, GSK, Pfizer, Advaxis, and Guardant Health; consulting/advisory role with Merck, BMS, Genentech, AstraZeneca, Amgen, Novartis, TPT, VI, Guardant Health; and honoraria from PER and OncLive, all outside the submitted work. JEH reports research funding from Regeneron and Dendreon; and travel, accommodations, and expenses from Genzyme. CH reports funding from the Henry Ford Cancer Institute supporting the current work; research funding to institution from Merck, Exelixis, Bayer, AstraZeneca, Genentech, Dendreon and Bausch; personal fees from Sanofi/Genzyme, Dendreon, Exelixis, Bristol Myers Squibb, Astellas, Medivation, Bayer, and Janssen Scientific, all outside the submitted work; and stock ownership by an immediate family member in Johnson and Johnson. DBJ reports advisory board participation for Array Biopharma, BMS, Catalyst Biopharma, Iovance, Jansen, Merck, Novartis, and OncoSec, and receives research funding from BMS and Incyte, all outside the submitted work. AK reports support to his institution from TESARO, Halozyme, Geistlich Pharma, Astellas Pharma, and Rafael Pharmaceuticals; and honoraria from OncLive, outside the submitted work. ARK (or an immediate family member) has currently or during the past 2 years owned stock or held an ownership interest in Merck, Sanofi, and BMS. VSK reports personal fees from Pfizer, Janssen, Dendreon, AstraZeneca, Seattle Genetics, and Clovis; grants (for institution) from Nektar, Novartis/Endocyte, Janssen, Clovis, and Prostate Cancer Foundation, all outside the submitted work. NMK reports personal fees from G1 Therapeutics, Invitae, Beyond Spring, Spectrum, BMS, Janssen, and Total Health, all outside the submitted work. PEL reports consulting/advisory role with Pfizer, Merck, Teva, BI, and Astra Zeneca, all outside the submitted work. AL-B reports personal fees from PSI CRO, Bayer, Blueprint, Astra-Zeneca, Medidata, Taiho, QED, Cardinal Health, BrightInsight, The Lynx Group, Boston Biomedical, Amgen, Bayer, Guardant, Natera, Eisai, Ipsen, and Merck; and stock options from Massive Bio, outside the submitted work. GdLL reports honoraria from Boehringer Ingelheim; consulting or advisory role for Pfizer and AstraZeneca; research funding from AstraZeneca; funding to his institution from Merck Sharp & Dohme, EMD Serono, AstraZeneca, Blueprint Medicines, Tesaro, Bavarian Nordic, NOVARTIS, G1 Therapeutics, Adaptimmune, BMS, GSK, AbbVie, Rgenix, Pfizer, Roche, Genentech, Lilly, and Janssen; travel, accommodations, and expenses from Boehringer Ingelheim, Pfizer, E.R. Squibb Sons, LLC, Janssen. GHL reports grants from AMGEN (institution); personal fees from G1 Therapeutics, TEVA, Samsung Bioepis, Beyond Spring, and Merck, outside the submitted work. RRM reports research funding from Bayer, Pfizer, Tempus; serves on Advisory Board for AstraZeneca, Bayer, Bristol Myers Squibb, Calithera, Exelixis, Janssen, Merck, Novartis, Pfizer, Sanofi, Tempus; is a consultant for Dendreon, Vividion; and serves on the molecular tumor board at Caris. RAM grants from Incyte, CTI, AbbVie, and Celgene; personal fees from Novartis, Genentech, Sierra Oncology, La Jolla, and Samus, outside the submitted work. VM has currently or during the past 2 years employment and stock or other ownership interest with Johnson & Johnson, all outside the submitted work. GN reports research funding to the institution from Novartis, all outside the submitted work. JN reports personal fees from AstraZeneca, Clovis Oncology; all outside the submitted work. CAP (or an immediate family member) has currently or during the past 2 years owned stock or held an ownership interest in Pfizer, Epizyme, Inovio, OPKO Health Inc, Roche. JMP reports grant from Dana-Farber/Harvard Cancer Center Breast SPORE Program, outside the submitted work. PP reports receiving payment for speakers' bureau from Novartis, Daichi Sankyo, Genentech, Seattle Genetics, and Pfizer, all outside the submitted work. NAP reports personal fees from Eli Lilly, Merck, BMS, Genentech, AstraZeneca, Inivata, and Regeneron, outside the submitted work. SP reports personal fees from AbbVie, Amgen, AstraZeneca, Bayer, Biocartis, Boehringer-Ingelheim, Bristol-Myers Squibb, Clovis, Daiichi Sankyo, Debiopharm, Eli Lilly, F. Hoffmann-La Roche, Foundation Medicine, Illumina, Janssen, Merck Sharp and Dohme, Merck Serono, Merrimack, Novartis, Pharma Mar, Pfizer, Regeneron, Sanofi, Seattle Genetics and Takeda, AstraZeneca, Boehringer-Ingelheim, Bristol-Myers Squibb, Eli Lilly, F. Hoffmann-La Roche, Merck Sharp and Dohme, Novartis, Pfizer, and Takeda; nonfinancial support from Amgen, AstraZeneca, Boehringer-Ingelheim, Bristol-Meyers Squibb, Clovis, F. Hoffmann-La Roche, Illumina, Merck Sharp and Dohme, Merck Serono, Novartis, Pfizer, and Sanofi; and personal fees from BioInvent (all fees to institution), outside the submitted work. DYR reports consulting/advisory role with and coverage of travel/accommodation expenses by Castle Biosciences, all outside the submitted work. BIR reports grants, personal fees, and nonfinancial support from Merck; grants and personal fees from BMS, Pfizer, Aveo, and Genentech; grants from Astra Zeneca; personal fees from Synthorx, 3D Medicines, Aravive, Surface Oncology, and Arrowhead Therapeutics; and other from PTC Therapeutics, outside the submitted work. RPR reports research grants to her institution from BMS and Janssen and has worked as a consultant/advisor and received honoraria from BMS and Janssen, all of which are outside the scope of submitted work. ALS reports travel support provided by Pfizer and Astellas. GKS reports personal fees from Apexigen, Array, Epizyme, GenCirq, Daiichi Sankyo, Fortress, Iovance Biotherapeutics, Bayer Pharmaceuticals, Pfizer Oncology, Array Advisory Board, Oncogenuity, Puretech, PTC Therapeutics, Ellipses Pharma, Concarlo; advisory board for Bionaut; grants from Astex; stock ownership in Pfizer, all outside the submitted work. SS reports stock and other ownership interests in Grand Rounds, Janssen, and Natera. YS reports honoraria from Boehringer Ingelheim, AstraZeneca, Novartis, and Eisai; consulting or advisory role with Pfizer, AstraZeneca, Novartis, Roche, Genentech, and Janssen, all outside the submitted work. MAT reports travel support from Syapse, Royalties from UpToDate, Connect MDS/AML Registry in Celgene (now owned by BMS), Myeloma Registry in Takeda; stock ownership in Doximity; personal fees from VIA Oncology (now owned by Elsevier ClinicalPath), Adaptive Advisory Board, and GSK; he is the local PI for Clinical Trials in AbbVie, BMS, CRAB CTC, Denovo, Research Network, Eli Lilly, LynxBio, Strata Oncology, and TG Therapeutics, all outside the submitted work. AKV reports research funding to the institution from BMS, MedPacto, Prelude, iOnctura, and Janssen; honoraria from Acceleron and Novartis; consulting/advisory role with Stelexis and Janssen; stock or other ownership in Stelexis; and an immediate family member with employment/leadership with CereXis, all outside the submitted work. DCV reports honoraria and speakers' bureau fees from CSL Behring, Merck Canada, Novartis Canada, Takeda, and UCB Biosciences GmbH, and travel accommodations from CSL Behring, and Avir Pharma, all outside the submitted work. He is supported by the Fonds de la recherche en santé du Québec (FRQS) Clinician-Scientist Junior 2 program. JLW reports grants from the National Cancer Institute during the conduct of the study; personal fees from Westat and IBM Watson Health; and other from HemOnc.org LLC, outside the submitted work. TMW-D reports stock and other ownership interests in High Enroll; honoraria from Physicians' Education Resource; consulting or advisory roles with Shattuck Labs, Rakuten Medical, Exicure; research funding from Merck, AstraZeneca/MedImmune, Bristol-Myers Squibb, GlaxoSmithKline, Caris Life Sciences, GlaxoSmithKline; travel, accommodations, expenses from Merck, Bristol-Myers Squibb, Bexion, AstraZeneca/MedImmune, Caris Life Sciences, Lilly, and Tesaro, all outside the submitted work. EW-B reports work in a consultant/advisor role for Astellas and BMS; funding support from Pfizer Global Medical Grants; other for Exelixis; and an immediate family member with stock ownership in Immunomedics and Nektar, all outside the submitted work. TZ reports research funding (to Duke) from Pfizer, Janssen, Acerta, AbbVie, Novartis, Merrimack, OmniSeq, PGDx, Merck, Mirati, Astellas, and Regeneron; consulting/speaking role with Genentech Roche, Exelixis, Genomic Health, and Sanofi Aventis; and serves on the consulting/advisory board for AstraZeneca, Bayer, Pfizer, Foundation Medicine, Janssen, Amgen, BMS, Calithera, Dendreon, and MJH Associates; stock ownership/employment (spouse) from Capio Biosciences, Archimmune Therapeutics, and Nanorobotics. AYZ has currently or during the past 2 years owned stock or held an ownership interest in Gilead Sciences. LZ reports personal fees from MERCK, outside the submitted work. All others have declared no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.

In human embryonic kidney (HEK) cells stably transfected with green fluorescent protein targeted to the endoplasmic reticulum (ER), elevation of intracellular Ca2+ ([Ca2+]i) altered ER morphology, making it appear punctate. Electron microscopy revealed that these punctate structures represented circular and branched rearrangements of the endoplasmic reticulum, but did not involve obvious swelling or pathological fragmentation. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA), prevented the effects of ionomycin on ER structure without affecting the elevation of [Ca2+]i. These results suggest that protein kinase C activation alters cytoplasmic or ER components underlying the effects of high [Ca2+]i on ER structure. Treatment of HEK cells with PMA also reduced the size of the thapsigargin-sensitive Ca2+ pool and inhibited Ca2+ entry in response to thapsigargin. Thus, protein kinase C activation has multiple actions on the calcium storage and signalling function of the endoplasmic reticulum in HEK cells: (1) reduced intracellular Ca2+ storage capacity, (2) inhibition of capacitative Ca2+ entry, and (3) protection of the endoplasmic reticulum against the effects of high [Ca2+]i.

In the phospholipase C signaling system, Ca(2+) is mobilized from intracellular stores by an action of inositol 1,4,5-trisphosphate. The depletion of intracellular calcium stores activates a calcium entry mechanism at the plasma membrane called capacitative calcium entry. The signal for activating the entry is unknown but likely involves either the generation or release, or both, from the endoplasmic reticulum of some diffusible signal. Recent research has focused on mammalian homologues of the Drosophila TRP protein as potential candidates for capacitative calcium entry channels. This review summarizes current knowledge about the nature of capacitative calcium entry signals, as well as the potential role of mammalian TRP proteins as capacitative calcium entry channel molecules.

Phosphatidylinositol-specific phospholipase C (PLC) is a family of enzymes that occupy a pivotal role in one of the largest classes of cellular signaling pathways known. Mammalian PLC enzymes have been divided into four major classes and a variety of subclasses based on their structural characteristics and immunological differences. There have been five invertebrate PLC-encoding genes cloned thus far and these fall within three of the four major classes used in categorizing mammalian PLC. Four of these invertebrate genes have been cloned from Drosophila melanogaster and one is from Artemia, a brine shrimp. Structural characteristics of the invertebrate enzymes include the presence of highly conserved Box X and Box Y domains found in major types of mammalian PLC as well as novel features. Two of the invertebrate PLC genes encode multiple splice-variant subtypes which is a newly emerging level of diversity observed in mammalian enzymes. Studies of the invertebrate PLCs have contributed to the identification of the physiological functions of individual isozymes. These identified roles include cellular processes such as phototransduction, olfaction, cell growth and differentiation.

Screening of a Drosophila genomic DNA library at reduced stringency hybridization conditions using a rat PLC alpha cDNA probe yielded a gene which encodes a member of the protein disulfide isomerase/PLC alpha family. The gene has been localized to band 74C on the left arm of the third chromosome and has been designated dpdi. Northern analysis shows that the dpdi gene encodes a transcript that is 2.3 kb in length and is present throughout development as well as in both heads and bodies of adults. The deduced dpdi protein is 496 amino acids in length and contains two domains exhibiting high similarity to thioredoxin, two regions that are similar to the hormone binding domain of human estrogen receptor, and a sequence of four amino acids (KDEL) at the C-terminus which has been described by others as being responsible for retention of proteins in the endoplasmic reticulum. Overall, dpdi contains a higher similarity to rat protein disulfide isomerase (53% identical) than to rat PLC alpha (30% identical). However, it is unclear whether dpdi functions in vivo as a PDI or as a PLC, or both. Drosophila, with its well characterized genetics and the ability to generate mutants in a gene that has been cloned, provides an excellent system in which to resolve this issue.

The rpa (receptor potential absent) mutation of the blowfly, Calliphora erythrocephala, reduces the light-evoked responses of photoreceptor cells and renders the fly blind. This phenotype is similar to the phenotype caused by norpA mutations in Drosophila which have been shown to occur within a gene encoding phospholipase C. In Western blots, norpA antiserum stains a protein in homogenates of wild-type Calliphora eye and head that is similar in molecular weight to the norpA protein. Very little staining of this protein is observed in similar homogenates of rpa mutant. Moreover, norpA antiserum strongly stains retina in immunohistochemical assays of wild-type adult head, but not in rpa mutant. Furthermore, eyes of rpa mutant have a reduced amount of phospholipase C activity compared to eye of wild-type Calliphora. These data suggest that the rpa mutation occurs in a phospholipase C gene of the blowfly that is homologous to the norpA gene of Drosophila.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. Molecular cloning of the norpA gene revealed that it encodes phosphatidylinositol-specific phospholipase C, which enzymes play a pivotal role in one of the largest classes of signaling pathways known. We have used Northern analysis, Western blots, phospholipase C activity assays, and immunohistochemical staining of tissues to examine the tissue-specific expression of the norpA gene and found that it is expressed in a variety of tissues besides the eye. Hybridization of norpA cRNA probe to blots of poly(A+) RNA reveals that the gene encodes at least four transcripts: a 7.5-kilobase (kb) transcript that is expressed in eye and 6.5-, 5.5-, and 5.0-kb transcripts that are expressed in adult body or early stages of development. Antiserum generated against the major gene product of norpA recognizes a 130-kDa protein that is abundant in eyes but severely reduced or absent in norpA mutants, a result which is consistent with previous conclusions that the norpA gene encodes an essential component of the visual system. However, the norpA antiserum also recognizes a 130-kDa protein in adult legs, thorax, and male abdomen, but not female abdomen. These localizations are supported by results of phospholipase C activity assays which show that the norpA mutation reduces phospholipase C activity in each of the tissues in which norpA protein can be detected. Furthermore, immunohistochemical staining of tissue sections with the norpA antiserum demonstrates that the norpA protein is abundant in the retina and ocelli and is present to a lesser extent in the brain and thoracic nervous system. Since some of the above mentioned tissues that express norpA (such as thorax, legs, and abdomen) have no known photoreceptor tissue, we conclude that the norpA gene product is also likely to have a role in signaling pathways other than phototransduction.