Antibodies are employed ubiquitously in biomedical sciences,including for diagnostics and therapeutics.One of the most important uses is for immunohistochemical(IHC)staining,a process that has been improving and evolv...Antibodies are employed ubiquitously in biomedical sciences,including for diagnostics and therapeutics.One of the most important uses is for immunohistochemical(IHC)staining,a process that has been improving and evolving over decades.IHC is useful when properly employed,yet misuse of the method is widespread and contributes to the“reproducibility crisis”in science.We report some of the common problems encountered with IHC assays,and direct readers to a wealth of literature documenting and providing some solutions to this problem.We also describe a series of vignettes that include our approach to analytical validation of antibodies and IHC assays that have facilitated a number of biological insights into prostate cancer and the refutation of a controversial association of a viral etiology in gliomas.We postulate that a great deal of the problem with lack of accuracy in IHC assays stems from the lack of awareness by researchers for the critical necessity for end-users to validate IHC antibodies and assays in their laboratories,regardless of manufacturer claims or past publications.We suggest that one reason for the pervasive lack of end-user validation for research antibodies is that researchers fail to realize that there are two general classes of antibodies employed in IHC.First,there are antibodies that are“clinical grade”reagents used by pathologists to help render diagnoses that influence patient treatment.Such diagnostic antibodies,which tend to be highly validated prior to clinical implementation,are in the vast minority(e.g.<500).The other main class of antibodies are“research grade”antibodies(now numbering>3800000),which are often not extensively validated prior to commercialization.Given increased awareness of the problem,both the United States,National Institutes of Health and some journals are requiring investigators to provide evidence of specificity of their antibody-based assays.展开更多
Neuroendocrine(NE)transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis.Up to date,even if patients at high risk of transformation can be identi...Neuroendocrine(NE)transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis.Up to date,even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53(TP53)and Retinoblastoma Transcriptional Corepressor 1(RB1)mutations in their tumors,no therapeutic strategies are available to prevent or delay histological transformation.Upregulation of the cell cycle kinase Cell Division Cycle 7(CDC7)occurred in tumors during the initial steps of NE transformation,already after TP53/RB1 co-inactivation,leading to induced sensitivity to the CDC7 inhibitor simurosertib.CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen(MYC),implicated in stemness and histological transformation.Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy,even in the presence of simurosertib.CDC7 inhibition also markedly extended response to standard cytotoxics(cisplatin,irinotecan)in lung and prostate small cell carcinoma models.These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity,as well as to effectively treat NE tumors de novo or after transformation.As simurosertib clinical efficacy trials are ongoing,this concept could be readily translated for patients at risk oftransformation.展开更多
基金This work was supported by the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Cancer Center Support Grant(NIH/NCI grant P30 CA006973)the NIH/NCI SPORE in prostate Cancer P50 CA058236,the NIH/NCI U01 CA196390+1 种基金The Patrick C.Walsh Prostate Cancer Research Fund,US Department of Defense Prostate Cancer Research Program(W81XWH-14-2-0182,Prostate Cancer Biorepository Network PCBN)the Prostate Cancer Foundation.
文摘Antibodies are employed ubiquitously in biomedical sciences,including for diagnostics and therapeutics.One of the most important uses is for immunohistochemical(IHC)staining,a process that has been improving and evolving over decades.IHC is useful when properly employed,yet misuse of the method is widespread and contributes to the“reproducibility crisis”in science.We report some of the common problems encountered with IHC assays,and direct readers to a wealth of literature documenting and providing some solutions to this problem.We also describe a series of vignettes that include our approach to analytical validation of antibodies and IHC assays that have facilitated a number of biological insights into prostate cancer and the refutation of a controversial association of a viral etiology in gliomas.We postulate that a great deal of the problem with lack of accuracy in IHC assays stems from the lack of awareness by researchers for the critical necessity for end-users to validate IHC antibodies and assays in their laboratories,regardless of manufacturer claims or past publications.We suggest that one reason for the pervasive lack of end-user validation for research antibodies is that researchers fail to realize that there are two general classes of antibodies employed in IHC.First,there are antibodies that are“clinical grade”reagents used by pathologists to help render diagnoses that influence patient treatment.Such diagnostic antibodies,which tend to be highly validated prior to clinical implementation,are in the vast minority(e.g.<500).The other main class of antibodies are“research grade”antibodies(now numbering>3800000),which are often not extensively validated prior to commercialization.Given increased awareness of the problem,both the United States,National Institutes of Health and some journals are requiring investigators to provide evidence of specificity of their antibody-based assays.
基金This study was supported by PO1 NIH PO1CA163227(Prostate Cancer Donor Program),NIH T32 CA1600001(to A.Q.V.),NCI R01 CA264078(to C.M.R.and,H.A.Y.)The Doris Duke Foundation(Grant 2021184)(to MCH),NCI P50 CA97186(to M.H.and C.M.),NCI R35 CA263816(to C.M.R.),NCI U24 CA213274(to C.M.R.),Yasuda Medical Foundation(to K.K.)+4 种基金the American Lung Association(to A.Q.V.)the Druckenmiller Center for Lung Cancer Research(to A.Q.V.,K.K.,and C.M.R.)This study was also supported by the Regional Ministry of Health and Consume of Andalucia RC-0004-2020(SMP)the Carlos II Health Institute through the projects"PI20/01109 and PI23/01679"(Co-funded by European Regional Development Fund/European Social Fund"A way to make Europe""nvesting in your future")(SMP).
文摘Neuroendocrine(NE)transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis.Up to date,even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53(TP53)and Retinoblastoma Transcriptional Corepressor 1(RB1)mutations in their tumors,no therapeutic strategies are available to prevent or delay histological transformation.Upregulation of the cell cycle kinase Cell Division Cycle 7(CDC7)occurred in tumors during the initial steps of NE transformation,already after TP53/RB1 co-inactivation,leading to induced sensitivity to the CDC7 inhibitor simurosertib.CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen(MYC),implicated in stemness and histological transformation.Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy,even in the presence of simurosertib.CDC7 inhibition also markedly extended response to standard cytotoxics(cisplatin,irinotecan)in lung and prostate small cell carcinoma models.These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity,as well as to effectively treat NE tumors de novo or after transformation.As simurosertib clinical efficacy trials are ongoing,this concept could be readily translated for patients at risk oftransformation.
