Publications by authors named "Roland Stein"

110 Publications

Heterogenous impairment of α cell function in type 2 diabetes is linked to cell maturation state.

Cell Metab 2022 02;34(2):256-268.e5

Department of Pharmacology, University of Alberta, Edmonton, AB T6G2R3, Canada; Alberta Diabetes Institute, University of Alberta, Edmonton, AB T6G2R3, Canada. Electronic address:

In diabetes, glucagon secretion from pancreatic α cells is dysregulated. The underlying mechanisms, and whether dysfunction occurs uniformly among cells, remain unclear. We examined α cells from human donors and mice using electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses α cell exocytosis by reducing P/Q-type Ca channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat feeding of mice, α cells shift toward a "β cell-like" electrophysiological profile in concert with indications of impaired identity. In human α cells we identified links between cell membrane properties and cell surface signaling receptors, mitochondrial respiratory chain complex assembly, and cell maturation. Cell-type classification using machine learning of electrophysiology data demonstrated a heterogenous loss of "electrophysiologic identity" in α cells from donors with type 2 diabetes. Indeed, a subset of α cells with impaired exocytosis is defined by an enrichment in progenitor and lineage markers and upregulation of an immature transcriptomic phenotype, suggesting important links between α cell maturation state and dysfunction.
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http://dx.doi.org/10.1016/j.cmet.2021.12.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8852281PMC
February 2022

Lipid Droplets' Role in the Regulation of β-Cell Function and β-Cell Demise in Type 2 Diabetes.

Endocrinology 2022 03;163(3)

Department of Internal Medicine Carver College of Medicine, Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, Iowa 52242, USA.

During development of type 2 diabetes (T2D), excessive nutritional load is thought to expose pancreatic islets to toxic effects of lipids and reduce β-cell function and mass. However, lipids also play a positive role in cellular metabolism and function. Thus, proper trafficking of lipids is critical for β cells to maximize the beneficial effects of these molecules while preventing their toxic effects. Lipid droplets (LDs) are organelles that play an important role in the storage and trafficking of lipids. In this review, we summarize the discovery of LDs in pancreatic β cells, LD lifecycle, and the effect of LD catabolism on β-cell insulin secretion. We discuss factors affecting LD formation such as age, cell type, species, and nutrient availability. We then outline published studies targeting critical LD regulators, primarily in rat and human β-cell models, to understand the molecular effect of LD formation and degradation on β-cell function and health. Furthermore, based on the abnormal LD accumulation observed in human T2D islets, we discuss the possible role of LDs during the development of β-cell failure in T2D. Current knowledge indicates that proper formation and clearance of LDs are critical to normal insulin secretion, endoplasmic reticulum homeostasis, and mitochondrial integrity in β cells. However, it remains unclear whether LDs positively or negatively affect human β-cell demise in T2D. Thus, we discuss possible research directions to address the knowledge gap regarding the role of LDs in β-cell failure.
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http://dx.doi.org/10.1210/endocr/bqac007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8826878PMC
March 2022

Sex-biased islet β cell dysfunction is caused by the MODY MAFA S64F variant by inducing premature aging and senescence in males.

Cell Rep 2021 10;37(2):109813

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA. Electronic address:

A heterozygous missense mutation of the islet β cell-enriched MAFA transcription factor (p.Ser64Phe [S64F]) is found in patients with adult-onset β cell dysfunction (diabetes or insulinomatosis), with men more prone to diabetes than women. This mutation engenders increased stability to the unstable MAFA protein. Here, we develop a S64F MafA mouse model to determine how β cell function is affected and find sex-dependent phenotypes. Heterozygous mutant males (MafA) display impaired glucose tolerance, while females are slightly hypoglycemic with improved blood glucose clearance. Only MafA males show transiently higher MafA protein levels preceding glucose intolerance and sex-dependent changes to genes involved in Ca signaling, DNA damage, aging, and senescence. MAFA production in male human β cells also accelerate cellular senescence and increase senescence-associated secretory proteins compared to cells expressing MAFA. These results implicate a conserved mechanism of accelerated islet aging and senescence in promoting diabetes in MAFA carriers in a sex-biased manner.
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http://dx.doi.org/10.1016/j.celrep.2021.109813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8845126PMC
October 2021

Identification of direct transcriptional targets of NFATC2 that promote β cell proliferation.

J Clin Invest 2021 11;131(21)

Biochemistry Department, University of Wisconsin-Madison, Madison, Wisconsin, USA.

The transcription factor NFATC2 induces β cell proliferation in mouse and human islets. However, the genomic targets that mediate these effects have not been identified. We expressed active forms of Nfatc2 and Nfatc1 in human islets. By integrating changes in gene expression with genomic binding sites for NFATC2, we identified approximately 2200 transcriptional targets of NFATC2. Genes induced by NFATC2 were enriched for transcripts that regulate the cell cycle and for DNA motifs associated with the transcription factor FOXP. Islets from an endocrine-specific Foxp1, Foxp2, and Foxp4 triple-knockout mouse were less responsive to NFATC2-induced β cell proliferation, suggesting the FOXP family works to regulate β cell proliferation in concert with NFATC2. NFATC2 induced β cell proliferation in both mouse and human islets, whereas NFATC1 did so only in human islets. Exploiting this species difference, we identified approximately 250 direct transcriptional targets of NFAT in human islets. This gene set enriches for cell cycle-associated transcripts and includes Nr4a1. Deletion of Nr4a1 reduced the capacity of NFATC2 to induce β cell proliferation, suggesting that much of the effect of NFATC2 occurs through its induction of Nr4a1. Integration of noncoding RNA expression, chromatin accessibility, and NFATC2 binding sites enabled us to identify NFATC2-dependent enhancer loci that mediate β cell proliferation.
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http://dx.doi.org/10.1172/JCI144833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8553569PMC
November 2021

Lipid Droplets Protect Human β-Cells From Lipotoxicity-Induced Stress and Cell Identity Changes.

Diabetes 2021 11 25;70(11):2595-2607. Epub 2021 Aug 25.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN

Free fatty acids (FFAs) are often stored in lipid droplet (LD) depots for eventual metabolic and/or synthetic use in many cell types, such a muscle, liver, and fat. In pancreatic islets, overt LD accumulation was detected in humans but not mice. LD buildup in islets was principally observed after roughly 11 years of age, increasing throughout adulthood under physiologic conditions, and also enriched in type 2 diabetes. To obtain insight into the role of LDs in human islet β-cell function, the levels of a key LD scaffold protein, perilipin 2 (PLIN2), were manipulated by lentiviral-mediated knockdown (KD) or overexpression (OE) in EndoCβH2-Cre cells, a human cell line with adult islet β-like properties. Glucose-stimulated insulin secretion was blunted in PLIN2KD cells and improved in PLIN2OE cells. An unbiased transcriptomic analysis revealed that limiting LD formation induced effectors of endoplasmic reticulum (ER) stress that compromised the expression of critical β-cell function and identity genes. These changes were essentially reversed by PLIN2OE or using the ER stress inhibitor, tauroursodeoxycholic acid. These results strongly suggest that LDs are essential for adult human islet β-cell activity by preserving FFA homeostasis.
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http://dx.doi.org/10.2337/db21-0261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8564404PMC
November 2021

Combinatorial transcription factor profiles predict mature and functional human islet α and β cells.

JCI Insight 2021 09 22;6(18). Epub 2021 Sep 22.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells, and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled more than 40,000 cells from normal human islets by single-cell RNA-Seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-Seq, MAFA/MAFB-coexpressing β cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.
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http://dx.doi.org/10.1172/jci.insight.151621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8492318PMC
September 2021

Decellularized Tissue Matrix Enhances Self-Assembly of Islet Organoids from Pluripotent Stem Cell Differentiation.

ACS Biomater Sci Eng 2020 07 11;6(7):4155-4165. Epub 2020 Jun 11.

Department of Biomedical Engineering, Binghamton University, State University of New York (SUNY), Binghamton, New York 13902, United States.

Regenerating human islet organoids from stem cells remains a significant challenge because of our limited knowledge on cues essential for developing the endocrine organoids in vitro. In this study, we discovered that a natural material prepared from a decellularized rat pancreatic extracellular matrix (dpECM) induces the self-assembly of human islet organoids during induced pluripotent stem cell (iPSC) pancreatic differentiation. For the first time, we demonstrated that the iPSC-derived islet organoids formed in the presence of the dpECM are capable of glucose-responsive secretion of both insulin and glucagon, two major hormones that maintain blood glucose homeostasis. The characterization of the organoids revealed that the organoids consisted of all major endocrine cell types, including α, β, δ, and pancreatic polypeptide cells, that were assembled into a tissue architecture similar to that of human islets. The exposure of iPSCs to the dpECM during differentiation resulted in considerably elevated expression of key pancreatic transcription factors such as PDX-1, MAFA, and NKX6.1 and the production of all major hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide from stem cell-derived organoids. This study highlights the importance of natural, bioactive biomaterials for building microenvironments crucial to regenerating islet organoids from stem cells.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00088DOI Listing
July 2020

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in β Cells.

Cell Metab 2020 12 13;32(6):1028-1040.e4. Epub 2020 Nov 13.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA; VA Tennessee Valley Healthcare System, Nashville, TN 37212, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

Isolated reports of new-onset diabetes in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2 is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into β cells via co-expression of its canonical cell entry factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2); however, their expression in human pancreas has not been clearly defined. We analyzed six transcriptional datasets of primary human islet cells and found that ACE2 and TMPRSS2 were not co-expressed in single β cells. In pancreatic sections, ACE2 and TMPRSS2 protein was not detected in β cells from donors with and without diabetes. Instead, ACE2 protein was expressed in islet and exocrine tissue microvasculature and in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. These findings reduce the likelihood that SARS-CoV-2 directly infects β cells in vivo through ACE2 and TMPRSS2.
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http://dx.doi.org/10.1016/j.cmet.2020.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664344PMC
December 2020

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 are Expressed in the Pancreas but are Not Enriched in Islet Endocrine Cells.

bioRxiv 2020 Oct 20. Epub 2020 Oct 20.

Reports of new-onset diabetes and diabetic ketoacidosis in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2, the virus that causes COVID-19, is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into host β cells via cell surface co-expression of ACE2 and TMPRSS2, the putative receptor and effector protease, respectively. To define ACE2 and TMPRSS2 expression in the human pancreas, we examined six transcriptional datasets from primary human islet cells and assessed protein expression by immunofluorescence in pancreata from donors with and without diabetes. and transcripts were low or undetectable in pancreatic islet endocrine cells as determined by bulk or single cell RNA sequencing, and neither protein was detected in α or β cells from these donors. Instead, ACE2 protein was expressed in the islet and exocrine tissue microvasculature and also found in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. The absence of significant ACE2 and TMPRSS2 co-expression in islet endocrine cells reduces the likelihood that SARS-CoV-2 directly infects pancreatic islet β cells through these cell entry proteins.
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http://dx.doi.org/10.1101/2020.08.31.275719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587777PMC
October 2020

In vivo studies of glucagon secretion by human islets transplanted in mice.

Nat Metab 2020 06 8;2(6):547-557. Epub 2020 Jun 8.

Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA.

Little is known about regulated glucagon secretion by human islet α-cells compared to insulin secretion from β-cells, despite conclusive evidence of dysfunction in both cell types in diabetes mellitus. Distinct insulins in humans and mice permit in vivo studies of human β-cell regulation after human islet transplantation in immunocompromised mice, whereas identical glucagon sequences prevent analogous in vivo measures of glucagon output from human α-cells. Here, we use CRISPR-Cas9 editing to remove glucagon codons 2-29 in immunocompromised NSG mice, preserving the production of other proglucagon-derived hormones. Glucagon knockout NSG (GKO-NSG) mice have metabolic, liver and pancreatic phenotypes associated with glucagon-signalling deficits that revert after transplantation of human islets from non-diabetic donors. Glucagon hypersecretion by transplanted islets from donors with type 2 diabetes revealed islet-intrinsic defects. We suggest that GKO-NSG mice provide an unprecedented resource to investigate human α-cell regulation in vivo.
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http://dx.doi.org/10.1038/s42255-020-0213-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7739959PMC
June 2020

Loss of the transcription factor MAFB limits β-cell derivation from human PSCs.

Nat Commun 2020 06 2;11(1):2742. Epub 2020 Jun 2.

UCSF Diabetes Center, University of California San Francisco, San Francisco, CA, 94143, USA.

Next generation sequencing studies have highlighted discrepancies in β-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human β-cells postnatally, while its expression is restricted to embryonic and neo-natal β-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to β-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide-positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology.
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http://dx.doi.org/10.1038/s41467-020-16550-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265500PMC
June 2020

Myt Transcription Factors Prevent Stress-Response Gene Overactivation to Enable Postnatal Pancreatic β Cell Proliferation, Function, and Survival.

Dev Cell 2020 05 30;53(4):390-405.e10. Epub 2020 Apr 30.

Vanderbilt Program in Developmental Biology, Department of Cell and Developmental Biology, and Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

Although cellular stress response is important for maintaining function and survival, overactivation of late-stage stress effectors cause dysfunction and death. We show that the myelin transcription factors (TFs) Myt1 (Nzf2), Myt2 (Myt1l, Nztf1, and Png-1), and Myt3 (St18 and Nzf3) prevent such overactivation in islet β cells. Thus, we found that co-inactivating the Myt TFs in mouse pancreatic progenitors compromised postnatal β cell function, proliferation, and survival, preceded by upregulation of late-stage stress-response genes activating transcription factors (e.g., Atf4) and heat-shock proteins (Hsps). Myt1 binds putative enhancers of Atf4 and Hsps, whose overexpression largely recapitulated the Myt-mutant phenotypes. Moreover, Myt(MYT)-TF levels were upregulated in mouse and human β cells during metabolic stress-induced compensation but downregulated in dysfunctional type 2 diabetic (T2D) human β cells. Lastly, MYT knockdown caused stress-gene overactivation and death in human EndoC-βH1 cells. These findings suggest that Myt TFs are essential restrictors of stress-response overactivity.
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http://dx.doi.org/10.1016/j.devcel.2020.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278035PMC
May 2020

Lipid Droplet Accumulation in Human Pancreatic Islets Is Dependent On Both Donor Age and Health.

Diabetes 2020 03 13;69(3):342-354. Epub 2019 Dec 13.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN

Human but not mouse islets transplanted into immunodeficient NSG mice effectively accumulate lipid droplets (LDs). Because chronic lipid exposure is associated with islet β-cell dysfunction, we investigated LD accumulation in the intact human and mouse pancreas over a range of ages and states of diabetes. Very few LDs were found in normal human juvenile pancreatic acinar and islet cells, with numbers subsequently increasing throughout adulthood. While accumulation appeared evenly distributed in postjuvenile acinar and islet cells in donors without diabetes, LDs were enriched in islet α- and β-cells from donors with type 2 diabetes (T2D). LDs were also found in the islet β-like cells produced from human embryonic cell-derived β-cell clusters. In contrast, LD accumulation was nearly undetectable in the adult rodent pancreas, even in hyperglycemic and hyperlipidemic models or 1.5-year-old mice. Taken together, there appear to be significant differences in pancreas islet cell lipid handling between species, and the human juvenile and adult cell populations. Moreover, our results suggest that LD enrichment could be impactful to T2D islet cell function.
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http://dx.doi.org/10.2337/db19-0281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034188PMC
March 2020

B Prognostic Score: External Validation of a Clinical Decision-making Tool for Metastatic Breast Cancer.

Clin Breast Cancer 2019 10 3;19(5):333-339. Epub 2019 May 3.

Department for Obstetrics and Gynecology, University of Würzburg Medical School, Würzburg, Germany.

Background: The B Prognostic Score (BPS) is a clinical decision-making tool in metastatic breast cancer (MBC) that provides risk classification based on routine parameters. This study validates the BPS in an independent series of MBC for the whole study group and for each intrinsic subtype.

Patients And Methods: We analyzed 641 metastasized patients, treated in 17 German certified breast cancer centers between 2001 and 2009. They were classified into low, intermediate, and high-risk groups according to BPS. Overall survival (OS) curves for the various BPS groups were compared with Kaplan-Meier method.

Results: According to the BPS formula, 42.3% of patients were classified as low risk, 25.4% as intermediate risk and 32.3% as high risk. Intermediate- and high-risk patients had a statistically significant decreased OS compared with BPS low-risk patients: (intermediate-risk: hazard ratio, 1.36; 95% confidence interval, 1.04-1.77; P = .023; high-risk: hazard ratio, 2.62; 95% confidence interval, 2.06-3.32; P < .001). The 5-year survival rates of low-, intermediate-, and high-risk patients were 41.3%, 26.9%, and 10.2%, respectively. The distribution of BPS risk groups varied significantly within the intrinsic subtypes. For each intrinsic subtype, BPS gives an additional risk classification.

Conclusions: This study demonstrates the reproducibility of the BPS based on routinely assessable parameters and confirms its prognostic value in an independent entire cohort of MBC as well as in the separate intrinsic subtypes. It therefore can help in counseling and individualizing the therapeutic regimens of those patients.
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http://dx.doi.org/10.1016/j.clbc.2019.04.015DOI Listing
October 2019

The Pdx1-Bound Swi/Snf Chromatin Remodeling Complex Regulates Pancreatic Progenitor Cell Proliferation and Mature Islet β-Cell Function.

Diabetes 2019 09 14;68(9):1806-1818. Epub 2019 Jun 14.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN

Transcription factors positively and/or negatively impact gene expression by recruiting coregulatory factors, which interact through protein-protein binding. Here we demonstrate that mouse pancreas size and islet β-cell function are controlled by the ATP-dependent Swi/Snf chromatin remodeling coregulatory complex that physically associates with Pdx1, a diabetes-linked transcription factor essential to pancreatic morphogenesis and adult islet cell function and maintenance. Early embryonic deletion of just the Swi/Snf Brg1 ATPase subunit reduced multipotent pancreatic progenitor cell proliferation and resulted in pancreas hypoplasia. In contrast, removal of both Swi/Snf ATPase subunits, Brg1 and Brm, was necessary to compromise adult islet β-cell activity, which included whole-animal glucose intolerance, hyperglycemia, and impaired insulin secretion. Notably, lineage-tracing analysis revealed Swi/Snf-deficient β-cells lost the ability to produce the mRNAs for and other key metabolic genes without effecting the expression of many essential islet-enriched transcription factors. Swi/Snf was necessary for Pdx1 to bind to the gene enhancer, demonstrating the importance of this association in mediating chromatin accessibility. These results illustrate how fundamental the Pdx1:Swi/Snf coregulator complex is in the pancreas, and we discuss how disrupting their association could influence type 1 and type 2 diabetes susceptibility.
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http://dx.doi.org/10.2337/db19-0349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702633PMC
September 2019

B lymphocytes protect islet β cells in diabetes prone NOD mice treated with imatinib.

JCI Insight 2019 04 9;5. Epub 2019 Apr 9.

Department of Pediatrics, Ian Burr Division of Endocrinology and Diabetes, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Imatinib (Gleevec) reverses type 1 diabetes (T1D) in NOD mice and is currently in clinical trials in individuals with recent-onset disease. While research has demonstrated that imatinib protects islet β cells from the harmful effects of ER stress, the role the immune system plays in its reversal of T1D has been less well understood, and specific cellular immune targets have not been identified. In this study, we demonstrate that B lymphocytes, an immune subset that normally drives diabetes pathology, are unexpectedly required for reversal of hyperglycemia in NOD mice treated with imatinib. In the presence of B lymphocytes, reversal was linked to an increase in serum insulin concentration, but not an increase in islet β cell mass or proliferation. However, improved β cell function was reflected by a partial recovery of MafA transcription factor expression, a sensitive marker of islet β cell stress that is important to adult β cell function. Imatinib treatment was found to increase the antioxidant capacity of B lymphocytes, improving reactive oxygen species (ROS) handling in NOD islets. This study reveals a novel mechanism through which imatinib enables B lymphocytes to orchestrate functional recovery of T1D β cells.
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http://dx.doi.org/10.1172/jci.insight.125317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538336PMC
April 2019

Imaging mass spectrometry enables molecular profiling of mouse and human pancreatic tissue.

Diabetologia 2019 06 6;62(6):1036-1047. Epub 2019 Apr 6.

9160 MRB III, Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232, USA.

Aims/hypothesis: The molecular response and function of pancreatic islet cells during metabolic stress is a complex process. The anatomical location and small size of pancreatic islets coupled with current methodological limitations have prevented the achievement of a complete, coherent picture of the role that lipids and proteins play in cellular processes under normal conditions and in diseased states. Herein, we describe the development of untargeted tissue imaging mass spectrometry (IMS) technologies for the study of in situ protein and, more specifically, lipid distributions in murine and human pancreases.

Methods: We developed matrix-assisted laser desorption/ionisation (MALDI) IMS protocols to study metabolite, lipid and protein distributions in mouse (wild-type and ob/ob mouse models) and human pancreases. IMS allows for the facile discrimination of chemically similar lipid and metabolite isoforms that cannot be distinguished using standard immunohistochemical techniques. Co-registration of MS images with immunofluorescence images acquired from serial tissue sections allowed accurate cross-registration of cell types. By acquiring immunofluorescence images first, this serial section approach guides targeted high spatial resolution IMS analyses (down to 15 μm) of regions of interest and leads to reduced time requirements for data acquisition.

Results: MALDI IMS enabled the molecular identification of specific phospholipid and glycolipid isoforms in pancreatic islets with intra-islet spatial resolution. This technology shows that subtle differences in the chemical structure of phospholipids can dramatically affect their distribution patterns and, presumably, cellular function within the islet and exocrine compartments of the pancreas (e.g. 18:1 vs 18:2 fatty acyl groups in phosphatidylcholine lipids). We also observed the localisation of specific GM3 ganglioside lipids [GM3(d34:1), GM3(d36:1), GM3(d38:1) and GM3(d40:1)] within murine islet cells that were correlated with a higher level of GM3 synthase as verified by immunostaining. However, in human pancreas, GM3 gangliosides were equally distributed in both the endocrine and exocrine tissue, with only one GM3 isoform showing islet-specific localisation.

Conclusions/interpretation: The development of more complete molecular profiles of pancreatic tissue will provide important insight into the molecular state of the pancreas during islet development, normal function, and diseased states. For example, this study demonstrates that these results can provide novel insight into the potential signalling mechanisms involving phospholipids and glycolipids that would be difficult to detect by targeted methods, and can help raise new hypotheses about the types of physiological control exerted on endocrine hormone-producing cells in islets. Importantly, the in situ measurements afforded by IMS do not require a priori knowledge of molecules of interest and are not susceptible to the limitations of immunohistochemistry, providing the opportunity for novel biomarker discovery. Notably, the presence of multiple GM3 isoforms in mouse islets and the differential localisation of lipids in human tissue underscore the important role these molecules play in regulating insulin modulation and suggest species, organ, and cell specificity. This approach demonstrates the importance of both high spatial resolution and high molecular specificity to accurately survey the molecular composition of complex, multi-functional tissues such as the pancreas.
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http://dx.doi.org/10.1007/s00125-019-4855-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553460PMC
June 2019

Cognate Nonlytic Interactions between CD8 T Cells and Breast Cancer Cells Induce Cancer Stem Cell-like Properties.

Cancer Res 2019 04 28;79(7):1507-1519. Epub 2019 Jan 28.

Department of Obstetrics and Gynecology, Würzburg University Hospital, University of Würzburg, Würzburg, Germany.

Targeting of tumor immune escape mechanisms holds enormous therapeutic potential. Still, most patients progress under immune checkpoint blockade and some even become hyperprogressors. To investigate how cancer cells respond to activated but ineffective T cells, we challenged peptide-loaded MCF-7 breast cancer cells with antigen-specific CD8 T cells in which lytic granules had been destroyed by pretreatment with Concanamycin A. Gene expression analysis after coculture revealed simultaneous induction of PD-L1, IDO1, CEACAM1, and further immunoregulatory checkpoints in breast cancer cells. Strikingly, we further observed gene signatures characteristic for dedifferentiation and acquisition of pluripotency markers including Yamanaka factors. Cognate interaction with nonlytic CD8 T cells also increased the proportion of stem cell-like cancer cells in a cell-to-cell contact- or (at least) proximity-dependent manner in various cell lines and in primary breast cancer cell cultures; this induction of stem cell-like properties was confirmed by enhanced tumor-forming capacity in immunodeficient mice. Resulting tumors were characterized by enhanced cell density, higher proliferation rates, and increased propensity for lymphoid metastasis. These findings describe a widely underappreciated pathway for immune escape, namely immune-mediated dedifferentiation of breast cancer cells, which is associated with profound changes in gene expression and cellular behavior. As the enhanced malignant potential of cancer cells after nonlytic cognate interactions with CD8 T cells enables increased tumor growth and metastasis in BALB/c mice, the described mechanism may provide a possible explanation for the clinical phenomenon of hyperprogression in response to unsuccessful immunotherapy. SIGNIFICANCE: This study shows that ineffective immune responses not only fail to clear a malignancy, but can also activate pathways in cancer cells that promote stemness and tumor-seeding capacity.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0387DOI Listing
April 2019

Human islets expressing HNF1A variant have defective β cell transcriptional regulatory networks.

J Clin Invest 2019 01 3;129(1):246-251. Epub 2018 Dec 3.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA.

Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained β cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.
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http://dx.doi.org/10.1172/JCI121994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307934PMC
January 2019

Examining How the MAFB Transcription Factor Affects Islet β-Cell Function Postnatally.

Diabetes 2019 02 13;68(2):337-348. Epub 2018 Nov 13.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN

The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell-enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express in adult mouse β-cells using transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal β-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet β-cells.
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http://dx.doi.org/10.2337/db18-0903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341297PMC
February 2019

Synaptotagmin 4 Regulates Pancreatic β Cell Maturation by Modulating the Ca Sensitivity of Insulin Secretion Vesicles.

Dev Cell 2018 05 12;45(3):347-361.e5. Epub 2018 Apr 12.

Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Department of Veterans Affairs, Tennessee Valley Health Authority, Nashville, TN 37232, USA; Center for Stem Cell Biology, Vanderbilt University School of Medicine, Department of Veterans Affairs, Tennessee Valley Health Authority, Nashville, TN 37232, USA; The Program of Developmental Biology, Vanderbilt University School of Medicine, Department of Veterans Affairs, Tennessee Valley Health Authority, Nashville, TN 37232, USA. Electronic address:

Islet β cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that β cells of newborns secrete more insulin than adults in response to similar intracellular Ca concentrations, suggesting differences in the Ca sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca binding paralog of the β cell Ca sensor Syt7, increased by ∼8-fold during β cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-βH1 cells, a human β cell line. These findings reveal the role that altered Ca sensing plays in regulating β cell maturation.
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http://dx.doi.org/10.1016/j.devcel.2018.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962294PMC
May 2018

Evaluation of sentinel lymph node biopsy prior to axillary lymph node dissection: the role of isolated tumor cells/micrometastases and multifocality/multicentricity-a retrospective study of 1214 breast cancer patients.

Arch Gynecol Obstet 2018 06 29;297(6):1509-1515. Epub 2018 Mar 29.

Department of Obstetrics and Gynecology, Klinikum Hanau, Academic Teaching Hospital of the Medical Faculty of the Goethe University of Frankfurt/Main, Leimenstraße 20, 63450, Hanau, Germany.

Purpose: Sentinel lymph node biopsy (SLNB) alone has thus become an accepted surgical approach for patients with limited axillary metastatic disease. We investigated to what extent isolated tumor cells (ITC) or micrometastasis in SLNBs is associated with proven tumor cells or metastasis in non-sentinel lymph nodes. Furthermore, we investigated the feasibility of SLNB in multifocal and multicentric tumors as both entities have been considered a contraindication for this technique.

Methods: 1214 women suffering from T1 and T2 invasive breast cancer, with clinically and sonographically insuspect axillary status and undergoing primary breast cancer surgery including SLNB and axillary staging in case of SLN (sentinel lymph node) metastases, were recruited into this multicentered study.

Results: ITC and micrometastases were found in 2.01 and 21.4% of patients with SLN metastases (n = 299). Among patients with sentinel micrometastases, 4.7% showed further axillary micrometastases, while only two patients (3.1%) had two axillary macrometastases. Multifocal and multicentric tumors were diagnosed in 9.3 and 2.6% of our patients who at least had one SLN resected, respectively. Detection rates of SLNs did not differ between the cohorts suffering from unicentric and multifocal or multicentric disease. Moreover, the portion of tumor-free SLNs, the number of SLNs with metastasis as well as the mean number of resected SLNs did not differ.

Conclusions: No patient with sentinel node micrometastases showed more than two axillary macrometastases. Multifocal and multicentric disease is no contraindication for SLNB.
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http://dx.doi.org/10.1007/s00404-018-4760-2DOI Listing
June 2018

α Cell Function and Gene Expression Are Compromised in Type 1 Diabetes.

Cell Rep 2018 03;22(10):2667-2676

Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Vanderbilt University Medical Center, Nashville, TN, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA; Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA. Electronic address:

Many patients with type 1 diabetes (T1D) have residual β cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual β cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant β cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and β cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-β cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.
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http://dx.doi.org/10.1016/j.celrep.2018.02.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368357PMC
March 2018

missense mutation causes familial insulinomatosis and diabetes mellitus.

Proc Natl Acad Sci U S A 2018 01 16;115(5):1027-1032. Epub 2018 Jan 16.

Institute of Biomedical and Clinical Science, University of Exeter Medical School, EX2 5DW Exeter, United Kingdom.

The β-cell-enriched MAFA transcription factor plays a central role in regulating glucose-stimulated insulin secretion while also demonstrating oncogenic transformation potential in vitro. No disease-causing variants have been previously described. We investigated a large pedigree with autosomal dominant inheritance of diabetes mellitus or insulinomatosis, an adult-onset condition of recurrent hyperinsulinemic hypoglycemia caused by multiple insulin-secreting neuroendocrine tumors of the pancreas. Using exome sequencing, we identified a missense mutation (p.Ser64Phe, c.191C>T) segregating with both phenotypes of insulinomatosis and diabetes. This mutation was also found in a second unrelated family with the same clinical phenotype, while no germline or somatic mutations were identified in nine patients with sporadic insulinomatosis. In the two families, insulinomatosis presented more frequently in females (eight females/two males) and diabetes more often in males (12 males/four females). Four patients from the index family, including two homozygotes, had a history of congenital cataract and/or glaucoma. The p.Ser64Phe mutation was found to impair phosphorylation within the transactivation domain of MAFA and profoundly increased MAFA protein stability under both high and low glucose concentrations in β-cell lines. In addition, the transactivation potential of p.Ser64Phe MAFA in β-cell lines was enhanced compared with wild-type MAFA. In summary, the p.Ser64Phe missense mutation leads to familial insulinomatosis or diabetes by impacting MAFA protein stability and transactivation ability. The human phenotypes associated with the p.Ser64Phe missense mutation reflect both the oncogenic capacity of MAFA and its key role in islet β-cell activity.
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http://dx.doi.org/10.1073/pnas.1712262115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798333PMC
January 2018

Evaluation of Sentinel Lymph Node Biopsy and Axillary Lymph Node Dissection for Breast Cancer Treatment Concepts - a Retrospective Study of 1,214 Breast Cancer Patients.

Breast Care (Basel) 2017 Oct 20;12(5):324-328. Epub 2017 Oct 20.

Department of Obstetrics and Gynecology, Hanau City Hospital, Hanau, Germany.

Background: Most breast cancer patients require lumpectomy with axillary sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND). The ACOSOG Z0011-trial failed to detect significant effects of ALND on disease-free and overall survival among patients with limited sentinel lymph node (SLN) metastases. Intense dose-dense chemotherapy and supraclavicular fossa radiation (SFR) are indicated for patients with extensive axillary metastases. In this multicentered study, we investigated the relevance of ALND after positive SLNB to determine adequate adjuvant therapy.

Methods: We retrospectively analyzed data from 1,214 patients with clinically nodal negative T1-T2 invasive breast cancer undergoing surgery at Hanau City Hospital Breast cancer center.

Results: 681 patients underwent ALND after SLNB. 20 patients (8.5%) from the group with 1 or 2 SLN metastases (n = 236) showed more than 3 lymph node metastases after ALND. 13 patients (31.7%) from the group with more than 2 SLN metastases (n = 41) were diagnosed with a minimum of 4 axillary lymph node metastases after ALND.

Conclusions: In 8.5% of the patients with 1 or 2 SLN metastases, ALND detected more than 3 macrometastases, setting the indication for intense dose-dense chemotherapy and SFR. More than 2 SLN metastases, T stage and grading predict lymph node metastases.
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http://dx.doi.org/10.1159/000477610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704708PMC
October 2017

Defining a Novel Role for the Pdx1 Transcription Factor in Islet β-Cell Maturation and Proliferation During Weaning.

Diabetes 2017 11 13;66(11):2830-2839. Epub 2017 Jul 13.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN

The transcription factor encoded by the gene is a critical transcriptional regulator, as it has fundamental actions in the formation of all pancreatic cell types, islet β-cell development, and adult islet β-cell function. Transgenic- and cell line-based experiments have identified 5'-flanking conserved sequences that control pancreatic and β-cell type-specific transcription, which are found within areas I (bp -2694 to -2561), II (bp -2139 to -1958), III (bp -1879 to -1799), and IV (bp -6200 to -5670). Because of the presence in area IV of binding sites for transcription factors associated with pancreas development and islet cell function, we analyzed how an endogenous deletion mutant affected expression embryonically and postnatally. The most striking result was observed in male mutant mice after 3 weeks of birth (i.e., the onset of weaning), with only a small effect on pancreas organogenesis and no deficiencies in their female counterparts. Compromised Pdx1 mRNA and protein levels in weaned male mutant β-cells were tightly linked with hyperglycemia, decreased β-cell proliferation, reduced β-cell area, and altered expression of Pdx1-bound genes that are important in β-cell replication, endoplasmic reticulum function, and mitochondrial activity. We discuss the impact of these novel findings to gene regulation and islet β-cell maturation postnatally.
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http://dx.doi.org/10.2337/db16-1516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652607PMC
November 2017

Interrupted Glucagon Signaling Reveals Hepatic α Cell Axis and Role for L-Glutamine in α Cell Proliferation.

Cell Metab 2017 Jun;25(6):1362-1373.e5

Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA; VA Tennessee Valley Healthcare, Nashville, TN 37212, USA. Electronic address:

Decreasing glucagon action lowers the blood glucose and may be useful therapeutically for diabetes. However, interrupted glucagon signaling leads to α cell proliferation. To identify postulated hepatic-derived circulating factor(s) responsible for α cell proliferation, we used transcriptomics/proteomics/metabolomics in three models of interrupted glucagon signaling and found that proliferation of mouse, zebrafish, and human α cells was mTOR and FoxP transcription factor dependent. Changes in hepatic amino acid (AA) catabolism gene expression predicted the observed increase in circulating AAs. Mimicking these AA levels stimulated α cell proliferation in a newly developed in vitro assay with L-glutamine being a critical AA. α cell expression of the AA transporter Slc38a5 was markedly increased in mice with interrupted glucagon signaling and played a role in α cell proliferation. These results indicate a hepatic α islet cell axis where glucagon regulates serum AA availability and AAs, especially L-glutamine, regulate α cell proliferation and mass via mTOR-dependent nutrient sensing.
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http://dx.doi.org/10.1016/j.cmet.2017.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572896PMC
June 2017

Evidence for Loss in Identity, De-Differentiation, and -Differentiation of Islet β-Cells in Type 2 Diabetes.

Front Genet 2017 29;8:35. Epub 2017 Mar 29.

Department of Molecular Physiology and Biophysics, Vanderbilt UniversityNashville, TN, USA.

The two main types of diabetes mellitus have distinct etiologies, yet a similar outcome: loss of islet β-cell function that is solely responsible for the secretion of the insulin hormone to reduce elevated plasma glucose toward euglycemic levels. Type 1 diabetes (T1D) has traditionally been characterized by autoimmune-mediated β-cell death leading to insulin-dependence, whereas type 2 diabetes (T2D) has hallmarks of peripheral insulin resistance, β-cell dysfunction, and cell death. However, a growing body of evidence suggests that, especially during T2D, key components of β-cell failure involves: (1) loss of cell identity, specifically proteins associated with mature cell function (e.g., insulin and transcription factors like MAFA, PDX1, and NKX6.1), as well as (2) de-differentiation, defined by regression to a progenitor or stem cell-like state. New technologies have allowed the field to compare islet cell characteristics from normal human donors to those under pathophysiological conditions by single cell RNA-Sequencing and through epigenetic analysis. This has revealed a remarkable level of heterogeneity among histologically defined "insulin-positive" β-cells. These results not only suggest that these β-cell subsets have different responses to insulin secretagogues, but that defining their unique gene expression and epigenetic modification profiles will offer opportunities to develop cellular therapeutics to enrich/maintain certain subsets for correcting pathological glucose levels. In this review, we will summarize the recent literature describing how β-cell heterogeneity and plasticity may be influenced in T2D, and various possible avenues of therapeutic intervention.
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http://dx.doi.org/10.3389/fgene.2017.00035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372778PMC
March 2017

Are There Breast Cancer Patients with Node-Negative Small Tumours, Who Do Not Benefit from Adjuvant Systemic Therapy?

Oncology 2017 24;92(6):317-324. Epub 2017 Mar 24.

Department for Obstetrics and Gynecology, University of Würzburg Medical School, Würzburg, Germany.

Objective: To identify subgroups of patients with pT1 pN0 breast cancer (BC) who might not profit from adjuvant systemic therapy (AST).

Methods: Data of 3,774 pT1 pN0 BC patients from 17 certified BC centres within the BRENDA study group were collected between 1992 and 2008 and retrospectively analysed. Uni- and multivariate analyses were performed using Kaplan-Meier methods and Cox regression models.

Results: 279 (7.4%) of the pT1 pN0 BC patients were T1a, 944 (25.0%) were T1b and 2,551 (67.6%) were T1c. There was no significant difference (p > 0.1) in recurrence-free survival (RFS)/overall survival (OAS) between patients with pT1a, pT1b, and T1c. Patients receiving any type of AST had a better outcome compared to women without AST after adjusting for age, tumour size, and intrinsic subtypes (RFS: p < 0.001; OAS: p < 0.001). AST was the most important prognostic parameter for RFS followed by intrinsic subtypes and age.

Conclusion: Patients with pT1 pN0 BC profit from AST independently of molecular subtypes, tumour size, age or comorbidity, with 5-year RFS of more than 95%. The correct definition of subgroups of patients who do not need AST is still an open question.
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http://dx.doi.org/10.1159/000455050DOI Listing
August 2017
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