Publications by authors named "Natasha Savage"

59 Publications

Engaging Pathology Residents in Scholarly Activities.

J Appl Lab Med 2021 Mar;6(2):567-569

Medical College of Georgia at Augusta University, GA.

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http://dx.doi.org/10.1093/jalm/jfaa094DOI Listing
March 2021

Engaging Pathology Residents in Clinical Chemistry: The Essential Ingredient Is a Committed Teacher.

J Appl Lab Med 2021 Mar;6(2):522-531

Department of Pathology, Medical College of Georgia at Augusta University, Augusta, GA.

Background: Pathology residents are thought to show a lack of interest in clinical chemistry, therefore potentially graduating from training programs unprepared to function as laboratory directors and clinical consultants.

Methods: A structured program of tutorials based primarily on Henry's textbook, supplemented by recent review articles; a question bank of about 600 questions to emphasize key concepts; requirement for performing and presenting quality improvement projects; participation in on-site CAP inspections; review of reference laboratory test requests; and involving residents in scholarly activity have resulted in sustained, transferable, and significant improvements in engagement, knowledge, competence, and examination scores.

Results: The primary parameter for measuring change in resident competence and engagement were improvements in resident in-service examination (RISE) scores, publications in peer-reviewed journals, and receipt of awards. The revised program produced significant improvement in RISE scores in clinical chemistry, over and above the improvements in the general residency program. The residents were authors on 12 publications in peer-reviewed PubMed listed journals in the 5-year period since revision in the clinical chemistry curriculum compared to no publications in clinical chemistry in the 5-year period before the new curriculum. Over the past 2 years, 6 of the 11 publications by graduating residents were in clinical chemistry, and 6 of 7 awards for research were garnered by residents engaged in clinical chemistry investigations. All of the residents passed their clinical pathology boards on first attempt since the change compared to 2 failures in the prior 5-year period.

Conclusions: The structured program described here is important as a template that could be adopted by any pathology training program. The question bank developed by this program is a valuable and transferable aid. However, success of such a program is dependent on the commitment of a knowledgeable, dedicated, and passionate teacher.
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http://dx.doi.org/10.1093/jalm/jfaa140DOI Listing
March 2021

Leukaemic relapse of anaplastic large cell lymphoma, ALK negative.

BMJ Case Rep 2021 Feb 22;14(2). Epub 2021 Feb 22.

Hematology Oncology, Augusta University, Augusta, Georgia, USA.

Anaplastic large cell lymphoma (ALCL), ALK negative (ALK-) is an aggressive lymphoproliferative disorder of mature T lymphocytes characterised by hallmark cells, CD30 positivity and lacking ALK protein expression. ALCL, ALK- has to be differentiated from peripheral T-cell lymphoma-not otherwise specified and classical Hodgkin's lymphoma. ALK- anaplastic large cell leukaemia should be considered in a patient with a history of ALCL, ALK- presenting with new leukaemia. We report a rare presentation of relapsed ALCL, ALK- with leukaemia after autologous stem cell transplantation in a 57-year-old male. Leukaemia, either as primary presentation or secondary transformation confers worse prognosis in ALCL, ALK- with very few cases reported so far. Emergency resuscitation with leukapheresis and treatment of tumour lysis syndrome along with supportive care should be followed by combination chemotherapy. Brentuximab vedotin and stem cell transplantation are the backbone of treatment for relapsed/refractory disease.
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http://dx.doi.org/10.1136/bcr-2020-239213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903072PMC
February 2021

Inclusion and Diversity in Pathology Residency Training.

J Appl Lab Med 2021 Jan;6(1):304-306

Departments of Pathology.

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http://dx.doi.org/10.1093/jalm/jfaa170DOI Listing
January 2021

Clinical performance and utility of a comprehensive next-generation sequencing DNA panel for the simultaneous analysis of variants, TMB and MSI for myeloid neoplasms.

PLoS One 2020 19;15(10):e0240976. Epub 2020 Oct 19.

Department of Pathology, Medical College of Georgia, Augusta University, Augusta, GA, United States of America.

The extensively employed limited-gene coverage NGS panels lead to clinically inadequate molecular profiling of myeloid neoplasms. The aim of the present investigation was to assess performance and clinical utility of a comprehensive DNA panel for myeloid neoplasms. Sixty-one previously well characterized samples were sequenced using TSO500 library preparation kit on NextSeq550 platform. Variants with a VAF ≥ 5% and a total read depth of >50X were filtered for analysis. The following results were recorded-for clinical samples: clinical sensitivity (97%), specificity (100%), precision (100%) and accuracy (99%) whereas reference control results were 100% for analytical sensitivity, specificity, precision and accuracy, with high intra- and inter-run reproducibility. The panel identified 880 variants across 292 genes, of which, 749 variants were in genes not covered in the 54 gene panel. The investigation revealed 14 variants in ten genes, and at least one was present in 96.2% patient samples that were pathogenic/ likely pathogenic in myeloid neoplasms. Also, 15 variants in five genes were found to be pathogenic/ likely pathogenic in other tumor types. Further, the TMB and MSI scores ranged from 0-7 and 0-9, respectively. The high analytical performance and clinical utility of this comprehensive NGS panel makes it practical and clinically relevant for adoption in clinical laboratories for routine molecular profiling of myeloid neoplasms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240976PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571681PMC
December 2020

DIAPH1 Mutation as a Novel Cause of Autosomal Dominant Macrothrombocytopenia and Hearing Loss.

Acta Haematol 2021 26;144(1):91-94. Epub 2020 Jun 26.

Division of Hematology/Oncology, Georgia Cancer Center, Augusta University, Augusta, Georgia, USA.

Macrothrombocytopenia (MTP) is a group of rare disorders characterized by giant platelets, thrombocytopenia, and variable association with abnormal bleeding. Inherited MTP are frequently misdiagnosed as immune thrombocytopenia. Associated second-organ manifestation can help narrow down syndromic MTPs. We describe a case of autosomal dominant sensorineural hearing loss and MTP caused by a gain of function mutation in DIAPH1. This mutation causes altered megarkaryopoiesis and platelet cytoskeletal deregulation. Although hearing loss and MTP were likely progressive, clinically significant bleeding was not observed. DIAPH1-related MTP can be distinguished clinically from MYH9 mutation by the absence of cataracts and glomerular disease.
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http://dx.doi.org/10.1159/000506727DOI Listing
March 2021

Epstein-Barr Virus-negative Marginal Zone Lymphoma as an Uncommon Form of Monomorphic Posttransplant Lymphoproliferative Disorder.

Am J Surg Pathol 2020 10;44(10):1340-1352

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD.

Monomorphic posttransplant lymphoproliferative disorders have been defined as lymphoid or plasmacytic proliferations that fulfill criteria for one of the B-cell or T/NK-cell neoplasms recognized in immunocompetent hosts in the current WHO Classification. Low-grade B-cell neoplasms have historically been excluded from this category, although rare reports of marginal zone lymphoma (MZL) have been described. We report 9 cases of posttransplant Epstein-Barr virus-negative MZL, all arising in solid organ transplant recipients (4 renal, 3 liver, 1 cardiac, and 1 liver, pancreas, and small bowel). Seven were extranodal MZL of mucosa-associated lymphoid tissue type, all of which had gastrointestinal involvement (4 colon, 1 duodenum, 1 stomach, and 1 oropharynx/base of tongue). Notably, the preferential involvement of intestine distinguishes posttransplant extranodal MZL from sporadic cases. Immunoglobulin light-chain restriction was seen in all cases, with polymerase chain reaction showing a monoclonal pattern in 7 of 8 cases with successful amplification of polymerase chain reaction products. A clonally unrelated recurrence was seen in one case. Next-generation sequencing identified recurrent mutations previously reported in MZL in 3/5 cases. MZL was diagnosed at least 1 year after solid organ transplant (median time to presentation, 84 mo; range, 13 to 108 mo). The median age was 44 (range, 9 to 73 y); the male: female ratio was 5:4. The mean follow-up was 33.4 months, with an indolent clinical course observed. A subset responded to reduction in immunosuppression and anti-CD20 therapy alone. These data support the designation of Epstein-Barr virus-negative MZL as an uncommon form of monomorphic posttransplant lymphoproliferative disorders.
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http://dx.doi.org/10.1097/PAS.0000000000001514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492423PMC
October 2020

Autoimmune Hemolytic Anemia in a Renal Transplant Patient Following Seasonal Influenza Vaccination.

Case Rep Hematol 2019 20;2019:3537418. Epub 2019 Oct 20.

Department of Pathology, Augusta University Health, Augusta, Georgia.

Vaccines aim to prevent disease occurrence, its severity, and resultant complications. Our patient, a 58-year-old male, received seasonal influenza vaccination as part of routine health maintenance. Three days later, he presented with malaise, fever, and yellowish discoloration of eyes. His labs showed hyperbilirubinemia, anemia, elevated lactate dehydrogenase, and low haptoglobin, consistent with hemolytic anemia. Autoimmune hemolytic anemia has been associated with vaccine use and may result from phenomena of molecular mimicry and cross-reactivity with the possible role of vaccine adjuvants as well. An underlying structural defect of the red blood cell membrane may make them prone to hemolysis. The differential diagnosis and work-up of hemolytic anemia is extensive, as performed in our case. Management strategies for vaccine-induced hemolysis may involve supportive care, red blood cell transfusion, steroids, and intravenous immunoglobulin.
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http://dx.doi.org/10.1155/2019/3537418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854248PMC
October 2019

Requiem for the STAT Test: Automation and Point of Care Testing.

Lab Med 2020 Mar;51(2):e27-e31

Administrative Director for Pathology Services, Medical College of Georgia at Augusta University, Department of Pathology, Augusta, Georgia.

Objective: Quick turnaround of laboratory test results is needed for medical and administrative reasons. Historically, laboratory tests have been requested as routine or STAT. With a few exceptions, a total turnaround time of 90 minutes has been the usually acceptable turnaround time for STAT tests.

Methods: We implemented front-end automation and autoverification and eliminated batch testing for routine tests. We instituted on-site intraoperative testing for selected analytes and employed point of care (POC) testing judiciously. The pneumatic tube system for specimen transport was expanded.

Results: The in-laboratory turnaround time was reduced to 45 minutes for more than 90% of tests that could reasonably be ordered STAT. With rare exceptions, the laboratory no longer differentiates between routine and STAT testing. Having a single queue for all tests has improved the efficiency of the laboratory.

Conclusion: It has been recognized in manufacturing that batch processing and having multiple queues for products are inefficient. The same principles were applied to laboratory testing, which resulted in improvement in operational efficiency and elimination of STAT tests. We propose that the target for in-laboratory turnaround time for STAT tests, if not all tests, be 45 minutes or less for more than 90% of specimens.
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http://dx.doi.org/10.1093/labmed/lmz080DOI Listing
March 2020

Dormant pathogenic CD4 T cells are prevalent in the peripheral repertoire of healthy mice.

Nat Commun 2019 10 25;10(1):4882. Epub 2019 Oct 25.

Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA.

Thymic central tolerance eliminates most immature T cells with autoreactive T cell receptors (TCR) that recognize self MHC/peptide complexes. Regardless, an unknown number of autoreactive CD4Foxp3 T cells escape negative selection and in the periphery require continuous suppression by CD4Foxp3 regulatory cells (Tregs). Here, we compare immune repertoires of Treg-deficient and Treg-sufficient mice to find Tregs continuously constraining one-third of mature CD4Foxp3 cells from converting to pathogenic effectors in healthy mice. These dormant pathogenic clones frequently express TCRs activatable by ubiquitous autoantigens presented by class II MHCs on conventional dendritic cells, including self-peptides that select them in the thymus. Our data thus suggest that identification of most potentially autoreactive CD4 T cells in the peripheral repertoire is critical to harness or redirect these cells for therapeutic advantage.
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http://dx.doi.org/10.1038/s41467-019-12820-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814812PMC
October 2019

Educational Case: Acute Promyelocytic Leukemia With .

Acad Pathol 2019 Jan-Dec;6:2374289519875647. Epub 2019 Sep 18.

Department of Pathology, Medical College of Georgia, Augusta University Medical Center, Augusta, GA, USA.

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http://dx.doi.org/10.1177/2374289519875647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753509PMC
September 2019

Tumefactive fibroinflammatory lesion successfully treated with Rituximab.

Intractable Rare Dis Res 2019 May;8(2):138-141

Department of Otolaryngology, Head and Neck Surgery, Medical College of Georgia at Augusta University, Augusta, GA, USA.

Skull base pseudotumors, or tumefactive fibroinflammatory lesions (TFIL), are tumors characterized by local destruction with benign histopathology. Treatment includes surgery and steroids with varying degrees of symptom relief. A 45-year-old female presented with right otorrhea and middle ear effusion, which progressed to CN V pain/numbness, trismus, headache, and autophony. MRI showed a diffuse infiltrating mass in the right infratemporal region involving the trigeminal ganglion. Biopsy revealed benign fibromuscular and adipose tissue with lymphoplasmacytic infiltrate, giving a diagnosis of TFIL. Resection would be very difficult given tumor location. Initial treatment included an extended course of steroids without response, and interval disease progression. Two courses of rituximab 375 mg/m weekly × 4 given 3 months apart were then completed with excellent tolerance. With sixteen months following induction, the patient reports minimal symptoms with radiographic findings confirming continued disease regression. Rituximab is a potential treatment option for patients with TFIL without response to steroids.
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http://dx.doi.org/10.5582/irdr.2019.01061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557232PMC
May 2019

Intraoperative Monitoring of Heparin: Comparison of Activated Coagulation Time and Whole Blood Heparin Measurements by Different Point-of-Care Devices with Heparin Concentration by Laboratory-Performed Plasma Anti-Xa Assay.

Lab Med 2019 Oct;50(4):348-356

Department of Pathology, Medical College of Georgia at Augusta University.

Background: Cardiac surgical interventions, extracorporeal membrane oxygenation, transcutaneous coronary-artery angioplasty, and stenting are carried out while patients are being treated with the anticoagulation drug heparin. Monitoring the level and reversal of heparinization during and at the conclusion of medical and surgical procedures is a critical issue in patient care.

Methods: We performed parallel testing of the ACCRIVA Hemochron Signature Elite ACT+ and Hemochron Response analyzer, iSTAT platform, and 2 Hepcon Hemostasis Management System (HMS) Plus analyzers for monitoring intraoperative heparin treatment. Laboratory anti-Xa assay was used as the criterion standard for heparin measurement.

Results: Poor correlation between the 2 Hemochron analyzers was identified at 0.78. Correlation between the analyzers on the i-STAT platform was 0.97. Regression analysis revealed that i-STAT values were generally lower, by 43 seconds, than Hemochron values. The correlation between Hepcon and i-STAT activated clotting time (ACT) results was 0.94. The i-STAT ACT results were generally 23 seconds lower than the Hepcon ACT values. Correlation coefficients on comparing Hepcon ACT and i-STAT ACT using laboratory anti-Xa assay were 0.83 and 0.87, respectively. The correlation between Hepcon heparin concentration and anti-Xa results was 0.85.

Conclusions: ACT monitoring with iSTAT offers good correlation between instruments and with the Hepcon ACT. Hepcon occupies a specific niche in cardiac operating departments because of its ability to provide additional information regarding heparin concentration; however, lack of suitable proficiency testing may impair its use. The iSTAT is a more reliable platform for broader, hospital-wide application.
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http://dx.doi.org/10.1093/labmed/lmz014DOI Listing
October 2019

Ufbp1 promotes plasma cell development and ER expansion by modulating distinct branches of UPR.

Nat Commun 2019 03 6;10(1):1084. Epub 2019 Mar 6.

Department of Biochemistry and Molecular Biology, Augusta University, Augusta, GA, 30912, USA.

The IRE1α/XBP1 branch of unfolded protein response (UPR) pathway has a critical function in endoplasmic reticulum (ER) expansion in plasma cells via unknown mechanisms; interestingly, another UPR branch, PERK, is suppressed during plasma cell development. Here we show that Ufbp1, a target and cofactor of the ufmylation pathway, promotes plasma cell development by suppressing the activation of PERK. By contrast, the IRE1α/XBP1 axis upregulates the expression of Ufbp1 and ufmylation pathway genes in plasma cells, while Ufbp1 deficiency impairs ER expansion in plasma cells and retards immunoglobulin production. Structure and function analysis suggests that lysine 267 of Ufbp1, the main lysine in Ufbp1 that undergoes ufmylation, is dispensable for the development of plasmablasts, but is required for immunoglobulin production and stimulation of ER expansion in IRE1α-deficient plasmablasts. Thus, Ufbp1 distinctly regulates different branches of UPR pathway to promote plasma cell development and function.
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http://dx.doi.org/10.1038/s41467-019-08908-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403283PMC
March 2019

SUV39H1 Represses the Expression of Cytotoxic T-Lymphocyte Effector Genes to Promote Colon Tumor Immune Evasion.

Cancer Immunol Res 2019 03 4;7(3):414-427. Epub 2019 Jan 4.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.

Despite the presence of CTLs in the tumor microenvironment, the majority of immunogenic human colon cancer does not respond to immune checkpoint inhibitor immunotherapy, and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs were present in both MSI and microsatellite stable colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 was significantly elevated in human colon carcinoma compared with normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment have high expression of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened on the basis of the SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain structure of the human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. On the basis of the structure of this small molecule, we modified it and chemically synthesized a small molecule, termed F5446, which has an EC of 0.496 μmol/L for SUV39H1 enzymatic activity. H3K9me3 was enriched in the promoters of , and in quiescent T cells. F5446 inhibited H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8 CTL-dependent manner Our data indicate that SUV39H1 represses CTL effector gene expression and, in doing so, confers colon cancer immune escape.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397681PMC
March 2019

Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis.

Cell Rep 2018 12;25(11):3036-3046.e6

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta University, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. Electronic address:

IL-10 functions as a suppressor of colitis and colitis-associated colon cancer, but it is also a risk locus associated with ulcerative colitis. The mechanism underlying the contrasting roles of IL-10 in inflammation and colon cancer is unknown. We report here that inflammation induces the accumulation of CD11bGr1 myeloid-derived suppressor cells (MDSCs) that express high levels of IL-10 in colon tissue. IL-10 induces the activation of STAT3 that directly binds to the Dnmt1 and Dnmt3b promoters to activate their expression, resulting in DNA hypermethylation at the Irf8 promoter to silence IRF8 expression in colon epithelial cells. Mice with Irf8 deleted in colonic epithelial cells exhibit significantly higher inflammation-induced tumor incidence. Human colorectal carcinomas have significantly higher DNMT1 and DNMT3b and lower IRF8 expression, and they exhibit significantly higher IRF8 promoter DNA methylation than normal colon. Our data identify the MDSC-IL-10-STAT3-DNMT3b-IRF8 pathway as a link between chronic inflammation and colon cancer initiation.
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http://dx.doi.org/10.1016/j.celrep.2018.11.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319669PMC
December 2018

How I investigate Eosinophilia.

Int J Lab Hematol 2019 Apr 30;41(2):153-161. Epub 2018 Nov 30.

Medical College of Georgia, Augusta University, Augusta, Georgia.

Eosinophilia is typically secondary, that is, reactive, in nature and is associated with a wide variety of neoplastic and non-neoplastic disorders. Clonal eosinophilia is also seen in a wide variety of hematopoietic neoplasms, and sub-classification can be diagnostically challenging. A proper evaluation of persistent eosinophilia involves correlation of clinical history, laboratory data, cellular morphology, and ancillary testing. Knowledge of appropriate ancillary testing is necessary for a timely diagnosis. We present a review of the literature regarding eosinophilia, including the 2016 World Health Organization (WHO) update of WHO-defined eosinophilic disorders. We also present a review of eosinophilia in a case-based format including guidelines for evaluation of both routine and challenging cases. The purpose of this guideline is not to provide an in-depth discussion of each diagnosis, but rather a practical method that all pathologists can utilize to investigate eosinophilia.
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http://dx.doi.org/10.1111/ijlh.12955DOI Listing
April 2019

Loss of Fas Expression and Function Is Coupled with Colon Cancer Resistance to Immune Checkpoint Inhibitor Immunotherapy.

Mol Cancer Res 2019 02 14;17(2):420-430. Epub 2018 Nov 14.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.

Despite the remarkable efficacy of immune checkpoint inhibitor (ICI) immunotherapy in various types of human cancers, colon cancer, except for the approximately 4% microsatellite-instable (MSI) colon cancer, does not respond to ICI immunotherapy. ICI acts through activating CTLs that use the Fas-FasL pathway as one of the two effector mechanisms to suppress tumor. Cancer stem cells are often associated with resistance to therapy including immunotherapy, but the functions of Fas in colon cancer apoptosis and colon cancer stem cells are currently conflicting and highly debated. We report here that decreased Fas expression is coupled with a subset of CD133CD24 colon cancer cells and . Consistent of the lower Fas expression level, this subset of CD133CD24Fas colon cancer cells exhibits decreased sensitivity to FasL-induced apoptosis. Furthermore, FasL selectively enriches CD133CD24Fas colon cancer cells. CD133CD24Fas colon cancer cells exhibit increased lung colonization potential in experimental metastatic mouse models and decreased sensitivity to tumor-specific CTL adoptive transfer and ICI immunotherapies. Interestingly, FasL challenge selectively enriched this subset of colon cancer cells in microsatellite-stable (MSS) but not in the MSI human colon cancer cell lines. Consistent with the downregulation of Fas expression in CD133CD24 cells, lower Fas expression level is significantly correlated with decreased survival in patients with human colon cancer. IMPLICATIONS: Our data determine that CD133CD24Fas colon cancer cells are capable to evade Fas-FasL cytotoxicity of tumor-reactive CTLs and targeting this subset of colon cancer cells is potentially an effective approach to suppress colon cancer immune evasion.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359951PMC
February 2019

Loss of the BCR-FGFR1 GEF Domain Suppresses RHOA Activation and Enhances B-Lymphomagenesis in Mice.

Cancer Res 2019 01 9;79(1):114-124. Epub 2018 Nov 9.

Georgia Cancer Center, Augusta, Georgia.

Transformation of hematopoietic stem cells by the BCR-FGFR1 fusion kinase found in a variant of stem cell leukemia/lymphoma (SCLL) syndrome leads to development of B-lymphomas in syngeneic mice and humans. In this study, we show that the relatively rapid onset of this leukemia is potentially related to oncogenic domains within the BCR component. BCR recruited a guanidine nucleotide exchange factor (GEF) domain to the fusion kinase to facilitate activation of small GTPases such as the Ras homology gene family, member A (RHOA). Deletion of this GEF domain increased leukemogenesis, enhanced cell survival and proliferation, and promoted stem cell expansion and lymph node metastasis. This suggests that, in an SCLL context, the presence of the endogenous GEF motif leads to reduced leukemogenesis. Indeed, loss of the GEF domain suppressed activation of RHOA and PTEN, leading to increased activation of AKT. Loss of the GEF domain enhanced cell proliferation and invasion potential, which was also observed in cells in which RHOA is knocked down, supported by the observation that overexpression of RHOA leads to reduced viability and invasion. depletion of RHOA in SCLL cells significantly increased disease progression and shortened latency. Collectively, these data show that the BCR GEF domain affects phenotypes associated with progression of SCLL through suppression of RHOA signaling. SIGNIFICANCE: RHOA activation is a critical event in the progression of BCR-FGFR1-driven leukemogenesis in stem cell leukemia and lymphoma syndrome and is regulated by the BCR GEF domain.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484845PMC
January 2019

Persistent indolent pancolonic marginal zone lymphoma of MALT-type with plasmacytic differentiation - A rare post-transplant lymphoma?

Hum Pathol (N Y) 2017 Nov 9;10:74-78. Epub 2017 Jul 9.

Center for Cancer Research, National Cancer Institute, Bethesda, MD, United States.

Marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is associated with chronic inflammatory disorders. We present an indolent pancolonic MALT lymphoma occurring in a 39-year-old female with history of autoimmune hepatitis requiring liver transplant in 1997 and ulcerative colitis diagnosed in 2004. Random biopsies from a grossly unremarkable surveillance colonoscopy in 2015 revealed a dense monomorphic plasmacytoid infiltrate causing expansion of lamina propria without significant crypt infiltration or destruction. These cells were positive for CD79a and CD138 and showed lambda restriction; however, CD20, CD43, CD56, HHV8, and EBER were negative. A similar pancolonic infiltrate was identified in all prior colorectal biopsies from 2010 and 2012 upon retrospective review. Subsequent computed tomography of the abdomen revealed no bowel wall thickening nor enlarged lymph nodes. Bone marrow revealed involvement consistent with stage IV disease. Biopsies from 2010 and 2015 demonstrated clonal immunoglobulin gene rearrangement. mutation was not detected. The overall features were indicative of MALT lymphoma. Although low-grade B-cell lymphomas are not considered part of the post-transplant lymphoproliferative disorder spectrum, such cases have been reported, and are typically EBV-negative. Patient underwent treatment with pentostatin for her MALT lymphoma reaching a sustained remission despite additional immunosuppression for resurgent hepatic dysfunction. To our knowledge, this is the first reported case of EBV-negative pancolonic MALT lymphoma with plasmacytic differentiation post liver transplant presenting in an indolent, asymptomatic fashion with persistence for greater than five years successfully managed without compromising the patient's liver transplant.
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http://dx.doi.org/10.1016/j.ehpc.2017.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019211PMC
November 2017

A novel immunohistochemical score to predict early mortality in acute myeloid leukemia patients based on indoleamine 2,3 dioxygenase expression.

Sci Rep 2017 10 16;7(1):12892. Epub 2017 Oct 16.

Department of Pathology, Medical College of Georgia at Augusta University, Augusta, USA.

Indoleamine 2,3 dioxygenase-1 (IDO-1) is an enzyme in the kynurenine pathway which augments tumor-induced immune tolerance. Previous studies in childhood acute myeloid leukemia (AML) have shown a negative correlation of IDO-1 mRNA expression with outcomes. The aim of our study was to develop a practical and objective immunohistochemical technique to quantify IDO-1 expression on diagnostic bone marrow biopsies of AML patients in order to facilitate its use in routine clinical practice. IDO-1 mRNA was extracted from diagnostic bone marrow specimens from 29 AML patients. IDO-1 protein expression was assessed in 40 cases via immunohistochemistry and quantified by a novel 'composite IDO-1 score'. In a univariate analysis, higher age (p = 0.0018), male gender (p = 0.019), high risk cytogenetics (p = 0.002), higher IDO-1 mRNA (p = 0.005), higher composite IDO-1 score (p < 0.0001) and not undergoing allogeneic stem cell transplant (SCT, p = 0.0005) predicted poor overall survival. In a multivariate model that included the aforementioned variables, higher composite IDO-1 score (p = 0.007) and not undergoing allogeneic SCT (p = 0.007) was found to significantly predict poor outcomes. Further, patients who failed induction had higher composite IDO-1 score (p = 0.01). In conclusion, 'composite IDO-1 score' is a prognostic tool that can help identify a certain subset of AML patients with 'early mortality'. This unique subset of patients can potentially benefit from specific IDO-1 inhibitor therapy, currently in clinical trials.
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http://dx.doi.org/10.1038/s41598-017-12940-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643528PMC
October 2017

Does Bernard-Soulier syndrome protect against thrombotic thrombocytopenic purpura?

J Clin Apher 2018 02 27;33(1):124-125. Epub 2017 Jun 27.

Department of Pathology, Augusta University Medical Center, Augusta, Georgia, 30912.

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http://dx.doi.org/10.1002/jca.21564DOI Listing
February 2018

JAK-STAT-mediated chronic inflammation impairs cytotoxic T lymphocyte activation to decrease anti-PD-1 immunotherapy efficacy in pancreatic cancer.

Oncoimmunology 2017;6(3):e1291106. Epub 2017 Feb 10.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, USA; Georgia Cancer Center, Augusta, GA, USA; Charlie Norwood VA Medical Center, Augusta, GA, USA.

Human pancreatic cancer does not respond to immune check point blockade immunotherapy. One key feature of pancreatic cancer is the association between its progression and chronic inflammation. Emerging evidence supports a key role for the JAK-STAT pathway in pancreatic cancer inflammation. We aimed at testing the hypothesis that sustained JAK-STAT signaling suppresses cytotoxic T lymphocyte (CTL) activation to counteract anti-PD-1 immunotherapy-induced CTL activity in pancreatic cancer. We show that human pancreatic carcinomas express high level of PD-L1 and exhibit low level of CTL infiltration. JAK-STAT inhibitor Ruxolitinib selectively inhibits STAT1 and STAT3 activation and increases CTL infiltration to induce a Tc1/Th1 immune response in the tumor microenvironment in an orthotopic pancreatic cancer mouse model. Ruxilitinib-mediated tumor suppressive efficacy diminishes in T-cell-deficient mice. Pancreatic tumor grows significantly faster in IFNγ-deficient mice. However, neutralizing IFNγ does not alter tumor growth but diminishes Ruxolitinib-induced tumor suppression , indicating that lymphocytes and IFNγ are essential for Ruxolitinib-induced host antitumor immune response. Both type I and type II interferons upregulate PD-L1 expression through the JAK-STAT signaling pathway in mouse pancreatic tumor cells. Tumor cells respond to activated T cells by activating STAT3. The inhibition of STAT3 downregulates immune suppressive cytokines production by tumor cells, resulting in increased T cell activation and effector function. Consequently, Ruxolitinib significantly improves the efficacy of anti-PD-1 immunotherapy. Our data demonstrate that Ruxolitinib is effective in the inhibition of systemic inflammation in the tumor microenvironment and therefore upregulates CTL infiltration and activation to overcome pancreatic cancer resistance to anti-PD-1 immunotherapy.
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http://dx.doi.org/10.1080/2162402X.2017.1291106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384417PMC
February 2017

The MLL1-H3K4me3 Axis-Mediated PD-L1 Expression and Pancreatic Cancer Immune Evasion.

J Natl Cancer Inst 2017 01 28;109(6). Epub 2017 Jan 28.

Affiliations of authors: Department of Biochemistry and Molecular Biology (CL, AVP, KL), Department of Pathology (NS), and Department of Biostatistics and Epidemiology (JLW), Medical College of Georgia, Augusta, GA; Georgia Cancer Center (CL, AVP, HS, KL) and Department of Biological Sciences (MES), Augusta University, Augusta, GA; Charlie Norwood VA Medical Center, Augusta, GA (CL, AVP, KL); Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC (NHO); Mycosynthetix, Inc., Hillsborough, NC (CP).

Background: Pancreatic cancer is one of the cancers where anti-PD-L1/PD-1 immunotherapy has been unsuccessful. What confers pancreatic cancer resistance to checkpoint immunotherapy is unknown. The aim of this study is to elucidate the underlying mechanism of PD-L1 expression regulation in the context of pancreatic cancer immune evasion.

Methods: Pancreatic cancer mouse models and human specimens were used to determine PD-L1 and PD-1 expression and cancer immune evasion. Histone methyltransferase inhibitors, RNAi, and overexpression were used to elucidate the underlying molecular mechanism of PD-L1 expression regulation. All statistical tests were two-sided.

Results: PD-L1 is expressed in 60% to 90% of tumor cells in human pancreatic carcinomas and in nine of 10 human pancreatic cancer cell lines. PD-1 is expressed in 51.2% to 52.1% of pancreatic tumor-infiltrating cytotoxic T lymphocytes (CTLs). Tumors grow statistically significantly faster in FasL-deficient mice than in wild-type mice (P = .03-.001) and when CTLs are neutralized (P = .03-<.001). H3K4 trimethylation (H3K4me3) is enriched in the cd274 promoter in pancreatic tumor cells. MLL1 directly binds to the cd274 promoter to catalyze H3K4me3 to activate PD-L1 transcription in tumor cells. Inhibition or silencing of MLL1 decreases the H3K4me3 level in the cd274 promoter and PD-L1 expression in tumor cells. Accordingly, inhibition of MLL1 in combination with anti-PD-L1 or anti-PD-1 antibody immunotherapy effectively suppresses pancreatic tumor growth in a FasL- and CTL-dependent manner.

Conclusions: The Fas-FasL/CTLs and the MLL1-H3K4me3-PD-L1 axis play contrasting roles in pancreatic cancer immune surveillance and evasion. Targeting the MLL1-H3K4me3 axis is an effective approach to enhance the efficacy of checkpoint immunotherapy against pancreatic cancer.
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http://dx.doi.org/10.1093/jnci/djw283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291187PMC
January 2017

The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells.

Oncoimmunology 2016;5(12):e1247135. Epub 2016 Oct 20.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, USA; Charlie Norwood VA Medical Center, Augusta, GA, USA; Georgia Cancer Center, Augusta University, Augusta, GA, USA.

Programmed death-ligand 1 (PD-L1) is an inhibitory ligand that binds to PD-1 to suppress T cell activation. PD-L1 is constitutively expressed and inducible in tumor cells, but the expression profiles and regulatory mechanism of PD-L1 in myeloid-derived suppressor cells (MDSCs) are largely unknown. We report that PD-L1 is abundantly expressed in tumor-infiltrating leukocytes in human patients with both microsatellite instable and microsatellite stable colon cancer. About 60% CD11bCD33HLA-DR MDSCs from peripheral blood of human colon cancer patients are PD-L1. PD-L1 MDSCs are also significantly higher in tumor-bearing mice than in tumor-free mice. Interestingly, the highest PD-L1 MDSCs were observed in the tumor microenvironment in which 56-71% tumor-infiltrating MDSCs are PD-L1. In contrast, PD-L1 MDSCs are significantly less in secondary lymphoid organs and peripheral blood as compared to the tumor tissues, whereas bone marrow MDSCs are essentially PD-L1 in tumor-bearing mice. IFNγ is highly expressed in cells of the tumor tissues and IFNγ neutralization significantly decreased PD-L1 MDSCs in the tumor microenvironment . However, IFNγ-activated pSTAT1 does not bind to the promoter in MDSCs. Instead, pSTAT1 activates expression of IRF1, IRF5, IRF7 and IRF8 in MDSCs, and only pSTAT1-activated IRF1 binds to a unique IRF-binding sequence element and chromatin in the promoter to activate PD-L1 transcription. Our data determine that PD-L1 is highly expressed in tumor-infiltrating MDSCs and in a lesser degree in lymphoid organs, and the pSTAT1-IRF1 axis regulates PD-L1 expression in MDSCs.
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http://dx.doi.org/10.1080/2162402X.2016.1247135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214087PMC
October 2016

Treatment of Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis with Lenalidomide in a Patient Testing Negative for 5q Deletion and V617F and W515K/L Mutations.

Hematol Rep 2016 Nov 2;8(4):6592. Epub 2016 Nov 2.

Department of Hematology/Oncology, Medical College of Georgia, Augusta University , Augusta, GA, USA.

Refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T) is a hematologic malignancy that often results in transfusion dependency and a hypercoagulable state. This rare disease currently lacks formal guidelines for treatment; however, various case reports have demonstrated efficacy in the use of lenalidomide. This immunomodulatory drug has shown promise in patients with 5q deletions, with reports of achieving transfusion independence and normalization of platelet counts. Herein we present the case of a 68-year-old African American woman with RARS-T who tested negative for 5q deletion and V617F and W515K/L mutations. Her treatment with lenalidomide therapy resulted in a five-year durable complete clinical response.
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http://dx.doi.org/10.4081/hr.2016.6592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5136742PMC
November 2016

Parallel Actin-Independent Recycling Pathways Polarize Cdc42 in Budding Yeast.

Curr Biol 2016 08 28;26(16):2114-26. Epub 2016 Jul 28.

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA. Electronic address:

The highly conserved Rho-family GTPase Cdc42 is an essential regulator of polarity in many different cell types. During polarity establishment, Cdc42 becomes concentrated at a cortical site, where it interacts with downstream effectors to orient the cytoskeleton along the front-back axis. To concentrate Cdc42, loss of Cdc42 by diffusion must be balanced by recycling to the front. In Saccharomyces cerevisiae, the guanine nucleotide dissociation inhibitor (GDI) Rdi1 recycles Cdc42 through the cytoplasm. Loss of Rdi1 slowed but did not eliminate Cdc42 accumulation at the front, suggesting the existence of other recycling pathways. One proposed pathway involves actin-directed trafficking of vesicles carrying Cdc42 to the front. However, we found no role for F-actin in Cdc42 concentration, even in rdi1Δ cells. Instead, Cdc42 was still able to exchange between the membrane and cytoplasm in rdi1Δ cells, albeit at a reduced rate. Membrane-cytoplasm exchange of GDP-Cdc42 was faster than that of GTP-Cdc42, and computational modeling indicated that such exchange would suffice to promote polarization. We also uncovered a novel role for the Cdc42-directed GTPase-activating protein (GAP) Bem2 in Cdc42 polarization. Bem2 was known to act in series with Rdi1 to promote recycling of Cdc42, but we found that rdi1Δ bem2Δ mutants were synthetically lethal, suggesting that they also act in parallel. We suggest that GAP activity cooperates with the GDI to counteract the dissipative effect of a previously unappreciated pathway whereby GTP-Cdc42 escapes from the polarity site through the cytoplasm.
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http://dx.doi.org/10.1016/j.cub.2016.06.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5956535PMC
August 2016

Development of ZMYM2-FGFR1 driven AML in human CD34+ cells in immunocompromised mice.

Int J Cancer 2016 08 4;139(4):836-40. Epub 2016 May 4.

Georgia Regents University Cancer Center, Augusta, GA.

Acute myelogenous leukemia (AML) has an overall poor survival rate and shows considerable molecular heterogeneity in its etiology. In the WHO classification there are >50 cytogenetic subgroups of AML, many showing highly specific chromosome translocations that lead to constitutive activation of individual kinases. In a rare stem cell leukemia/lymphoma syndrome, translocations involving 8p11 lead to constitutive activation of the fibroblast growth factor receptor 1 (FGFR1) kinase. This disorder shows myeloproliferative disease with almost invariable progresses to AML and conventional therapeutic strategies are largely unsuccessful. Because of the rare nature of this syndrome, models that faithfully recapitulate the human disease are needed to evaluate therapeutic strategies. The t(8;13)(p11;q12) chromosome translocation is most common rearrangement seen in this syndrome and creates a ZMYM2-FGFR1 chimeric kinase. To understand more about the molecular etiology of AML induced by this particular rearrangement, we have created a model human CD34+ cells transplanted into immunocompromized mice which develop myeloproliferative disease that progresses to AML with a long (>12 months) latency period. As in humans, these mice show hepatospenomegaly, hypercellular bone marrow and a CD45 + CD34 + CD13+ immunophenotype. Molecular studies demonstrate upregulation of genes such as KLF4 and FLT3 that promote stemness, and overexpression of MYC, which is associated with suppression of myeloid cell differentiation. This murine model, therefore, provides an opportunity to develop therapeutic strategies against the most common subtype within these FGFR1 driven neoplasms and study the molecular etiology in more depth.
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http://dx.doi.org/10.1002/ijc.30100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754922PMC
August 2016

Superresolution microscopy reveals a dynamic picture of cell polarity maintenance during directional growth.

Sci Adv 2015 Nov 13;1(10):e1500947. Epub 2015 Nov 13.

Department of Microbiology, Institute for Applied Biosciences, KIT, 76187 Karlsruhe, Germany. ; Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.

Polar (directional) cell growth, a key cellular mechanism shared among a wide range of species, relies on targeted insertion of new material at specific locations of the plasma membrane. How these cell polarity sites are stably maintained during massive membrane insertion has remained elusive. Conventional live-cell optical microscopy fails to visualize polarity site formation in the crowded cell membrane environment because of its limited resolution. We have used advanced live-cell imaging techniques to directly observe the localization, assembly, and disassembly processes of cell polarity sites with high spatiotemporal resolution in a rapidly growing filamentous fungus, Aspergillus nidulans. We show that the membrane-associated polarity site marker TeaR is transported on microtubules along with secretory vesicles and forms a protein cluster at that point of the apical membrane where the plus end of the microtubule touches. There, a small patch of membrane is added through exocytosis, and the TeaR cluster gets quickly dispersed over the membrane. There is an incessant disassembly and reassembly of polarity sites at the growth zone, and each new polarity site locus is slightly offset from preceding ones. On the basis of our imaging results and computational modeling, we propose a transient polarity model that explains how cell polarity is stably maintained during highly active directional growth.
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http://dx.doi.org/10.1126/sciadv.1500947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673053PMC
November 2015

Role of competition between polarity sites in establishing a unique front.

Elife 2015 Nov 2;4. Epub 2015 Nov 2.

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, United States.

Polarity establishment in many cells is thought to occur via positive feedback that reinforces even tiny asymmetries in polarity protein distribution. Cdc42 and related GTPases are activated and accumulate in a patch of the cortex that defines the front of the cell. Positive feedback enables spontaneous polarization triggered by stochastic fluctuations, but as such fluctuations can occur at multiple locations, how do cells ensure that they make only one front? In polarizing cells of the model yeast Saccharomyces cerevisiae, positive feedback can trigger growth of several Cdc42 clusters at the same time, but this multi-cluster stage rapidly evolves to a single-cluster state, which then promotes bud emergence. By manipulating polarity protein dynamics, we show that resolution of multi-cluster intermediates occurs through a greedy competition between clusters to recruit and retain polarity proteins from a shared intracellular pool.
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http://dx.doi.org/10.7554/eLife.11611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728132PMC
November 2015