Publications by authors named "Adnan Mansoor"

49 Publications

Subtype-specific and co-occurring genetic alterations in B-cell non-Hodgkin lymphoma.

Haematologica 2021 Apr 1. Epub 2021 Apr 1.

Section of Hematology, Department of Medicine, University of Calgary, Calgary, AB.

B-cell non-Hodgkin's lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL such as diffuse large B-cell lymphoma (DLBCL) have been comprehensively interrogated at the genomic level. But rarer subtypes such as mantle cell lymphoma (MCL) remain sparsely characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth; including DLBCL, MCL, follicular lymphoma (FL), and Burkitt lymphoma (BL). We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumor from each subtype, including the frequent genetic deregulation of the ubiquitin proteasome system (UPS). In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.
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http://dx.doi.org/10.3324/haematol.2020.274258DOI Listing
April 2021

Gene Expression Analysis of Pediatric Acute Myeloid Leukemia Identified a Hyperactive ASXL1/BAP1 Axis Linked with Poor Prognosis and over Expressed Epigenetic Modifiers.

Pediatr Hematol Oncol 2021 Sep 25;38(6):581-592. Epub 2021 Mar 25.

Department of Pathology and Laboratory Medicine, University of Calgary/Alberta Precision Laboratories (APL), Alberta, Canada.

Genetic aberrations in the epigenome are rare in pediatric AML, hence expression data in epigenetic regulation and its downstream effect is lacking in childhood AML. Our pilot study screened epigenetic modifiers and its related oncogenic signal transduction pathways concerning clinical outcomes in a small cohort of pediatric AML in KSA. RNA from diagnostic BM biopsies (n = 35) was subjected to expression analysis employing the nCounter Pan-Cancer pathway panel. The patients were dichotomized into low ASXL1 (17/35; 49%) and high ASXL1 (18/35; 51%) groups based on ROC curve analysis. Age, gender, hematological data or molecular risk factors (FLT3 mutation/molecular fusion) exposed no significant differences across these two distinct ASXL1 expression groups (P > 0.05). High ASXL1 expression showed linkage with high expression of other epigenetic modifiers (TET2/EZH2/IDH1&2). Our data showed that high ASXL1 mRNA is interrelated with increased BRCA1 associated protein-1 (BAP1) and its target gene E2F Transcription Factor 1 (E2F1) expression. High ASXL1 expression was associated with high mortality {10/18 (56%) vs. 1/17; (6%) P < 0 .002}. Low ASXL1 expressers showed better OS {740 days vs. 579 days; log-rank P= < 0.023; HR 7.54 (0.98-54.1)}. The association between high ASXL1 expression and epigenetic modifiers is interesting but unexplained and require further investigation. High ASXL1 expression is associated with BAP1 and its target genes. Patients with high ASXL1 expression showed poor OS without any association with a conventional molecular prognostic marker.
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http://dx.doi.org/10.1080/08880018.2021.1901808DOI Listing
September 2021

KLF1/EKLF expression in acute leukemia is correlated with chromosomal abnormalities.

Blood Cells Mol Dis 2020 07 13;83:102434. Epub 2020 Apr 13.

Department of Cell, Developmental, & Regenerative Biology, Black Family Stem Cell Institute, Tisch Cancer Institute, Mount Sinai School of Medicine, New York, NY, USA. Electronic address:

KLF1 (EKLF) is a master regulator of erythropoiesis and controls expression of a wide array of target genes. We interrogated human tissue microarray samples via immunohistological analysis to address whether levels of KLF1 protein are associated with leukemia. We have made the unexpected findings that higher KLF1 levels are correlated with cells containing abnormal chromosomes, and that high KLF1 expression is not limited to acute myeloid leukemia (AML) associated with erythroid/megakaryoblastic differentiation. Expression of KLF1 is associated with poor survival. Further analyses reveal that KLF1 directly regulates a number of genes that play a role in chromosomal integrity. Together these results suggest that monitoring KLF1 levels may provide a new marker for risk stratification and prognosis in patients with AML.
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http://dx.doi.org/10.1016/j.bcmd.2020.102434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269198PMC
July 2020

Fit-For-Purpose PD-L1 Biomarker Testing For Patient Selection in Immuno-Oncology: Guidelines For Clinical Laboratories From the Canadian Association of Pathologists-Association Canadienne Des Pathologistes (CAP-ACP).

Appl Immunohistochem Mol Morphol 2019 Nov/Dec;27(10):699-714

Canadian Immunohistochemistry Quality Control.

Since 2014, programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) checkpoint inhibitors have been approved by various regulatory agencies for the treatment of multiple cancers including melanoma, lung cancer, urothelial carcinoma, renal cell carcinoma, head and neck cancer, classical Hodgkin lymphoma, colorectal cancer, gastroesophageal cancer, hepatocellular cancer, and other solid tumors. Of these approved drug/disease combinations, a subset also has regulatory agency-approved, commercially available companion/complementary diagnostic assays that were clinically validated using data from their corresponding clinical trials. The objective of this document is to provide evidence-based guidance to assist clinical laboratories in establishing fit-for-purpose PD-L1 biomarker assays that can accurately identify patients with specific tumor types who may respond to specific approved immuno-oncology therapies targeting the PD-1/PD-L1 checkpoint. These recommendations are issued as 38 Guideline Statements that address (i) assay development for surgical pathology and cytopathology specimens, (ii) reporting elements, and (iii) quality assurance (including validation/verification, internal quality assurance, and external quality assurance). The intent of this work is to provide recommendations that are relevant to any tumor type, are universally applicable and can be implemented by any clinical immunohistochemistry laboratory performing predictive PD-L1 immunohistochemistry testing.
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http://dx.doi.org/10.1097/PAI.0000000000000800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887625PMC
July 2020

Expression Profiling Reveals Involvement of WNT Pathway in the Malignant Progression of Sessile Serrated Adenomas.

Am J Pathol 2019 09 11;189(9):1732-1743. Epub 2019 Jun 11.

Department of Pathology, Cumming School of Medicine and Alberta Public Laboratories, University of Calgary, Calgary, Alberta, Canada; Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada. Electronic address:

Approximately 15% to 20% of colorectal cancers are developed through the serrated pathway of tumorigenesis, which is associated with BRAF mutation, CpG island methylation phenotype, and MLH1 methylation. However, the detailed process of progression from sessile serrated adenoma (SSA) to dysplasia and carcinoma has not been elucidated. To further characterize mechanisms involved in the dysplastic progression of SSA, we investigated differential expressions of mRNAs between areas with and without dysplasia within the same SSA polyps. Significantly dysregulated genes in paired samples were applied for functional annotation and biological significance. The same lysates from a subset of matched samples were subjected for miRNA expression profiling. Differentially expressed miRNAs were determined, and their targeted mRNAs were compared in parallel to the list of differentially expressed mRNAs from an RNA sequencing study. Fourteen common mRNA targets were identified, which include AXIN2, a known indicator of WNT/β-catenin pathway activation. Together, in this study, different genes, pathways, and biological processes involved in the initiation and progression of dysplasia in the serrated pathway are documented. One of the most significant findings is the involvement of the WNT/β-catenin pathway in the dysplastic progression of SSAs with different genes being targeted in early versus advanced dysplasia.
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http://dx.doi.org/10.1016/j.ajpath.2019.05.009DOI Listing
September 2019

Diffuse large B-cell lymphoma in Southeast Asian cohort: expression patterns of B-cell receptor (BCR) repertoire and its linkage with molecular subtypes and response to R-CHOP therapy.

J Clin Pathol 2019 Sep 12;72(9):630-635. Epub 2019 Jun 12.

Pathology and Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada

Aims: Heightened B-cell receptor (BCR) activity in diffuse large B-cell lymphoma (DLBCL) is well established, and a subset of patients with relapsed DLBCL can benefit from BCR-targeted therapies. Universal outreach of such emerging therapies mandates forming a global landscape of BCR molecular signalling in DLBCL, including Southeast Asia.

Methods: 79 patients with DLBCL (nodal, 59% and extranodal, 41%) treated with rituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) therapy were selected. Expression levels of BCR and linked signalling pathway molecules were inter-related with Lymph2Cx-based cell of origin (COO) types and overall survival (OS).

Results: Activated B-cell (ABC) type DLBCL constituted 49% (39/79) compared with germinal centre B-cell (GCB) type DLBCL (29/79; 37%) and revealed poor prognosis (p=0.013). In ABC-DLBCL, high expression exerted poor response to R-CHOP, while OS in ABC-DLBCL with low expression was similar to GCB-DLBCL subtype (p=0.004). High expression coupled with a poor OS for ABC-DLBCL as well as GCB-DLBCL subtypes (p=0.001). Furthermore, high coexpression of ( / ) showed poor OS (p=0.019), which linked with upregulation of several genes associated with BCR repertoire and nuclear factor-kappa B pathway (p<0.01). In multivariate analysis, high and expression retained prognostic significance against established clinical predictive factors such as age, International Prognostic Index and COO (p<0.05).

Conclusions: Our data provide a clear association between high BCR activity in DLBCL and response to therapy in a distinct population. Molecular data provided here will pave the pathway for the provision of promising novel-targeted therapies to patients with DLBCL in Southeast Asia.
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http://dx.doi.org/10.1136/jclinpath-2019-205837DOI Listing
September 2019

A bioclinical prognostic model using MYC and BCL2 predicts outcome in relapsed/refractory diffuse large B-cell lymphoma.

Haematologica 2018 02 2;103(2):288-296. Epub 2017 Nov 2.

University of Calgary, AB, Canada

The objective of this study was to create a bioclinical model, based on clinical and molecular predictors of event-free and overall survival for relapsed/refractory diffuse large B-cell lymphoma patients treated on the Canadian Cancer Trials Group (CCTG) LY12 prospective study. In 91 cases, sufficient histologic material was available to create tissue microarrays and perform immunohistochemistry staining for CD10, BCL6, MUM1/IRF4, FOXP1, LMO2, BCL2, MYC, P53 and phosphoSTAT3 (pySTAT3) expression. Sixty-seven cases had material sufficient for fluorescent hybridization (FISH) for and In addition, 97 formalin-fixed, paraffin-embedded tissue samples underwent digital gene expression profiling (GEP) to evaluate , and expression, and to determine cell-of-origin (COO) using the Lymph2Cx assay. No method of determining COO predicted event-free survival (EFS) or overall survival (OS). Factors independently associated with survival outcomes in multivariate analysis included primary refractory disease, elevated serum lactate dehydrogenase (LDH) at relapse, and MYC or BCL2 protein or gene expression. A bioclinical score using these four factors predicted outcome with 3-year EFS for cases with 0-1 2-4 factors of 55% 16% (<0.0001), respectively, assessing MYC and BCL2 by immunohistochemistry, 46% vs. 5% (<0.0001) assessing and messenger ribonucleic acid (mRNA) by digital gene expression, and 42% 21% (=0.079) assessing and by FISH. This proposed bioclinical model should be further studied and validated in other datasets, but may discriminate relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients who could benefit from conventional salvage therapy from others who require novel approaches. The LY12 study; .
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http://dx.doi.org/10.3324/haematol.2017.179309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792273PMC
February 2018

Comparison of protein-based cell-of-origin classification to the Lymph2Cx RNA assay in a cohort of diffuse large B-cell lymphomas in Malaysia.

J Clin Pathol 2018 Mar 3;71(3):215-220. Epub 2017 Aug 3.

Division of Hematology and Transfusion Medicine, Department of Pathology and Laboratory Medicine, University of Calgary/Calgary Laboratory Services (CLS), Calgary, Canada.

Aims: The cell of origin (COO) based molecular characterisation into germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) subtypes are central to the pathogenesis and clinical course in diffuse large B-cell lymphoma (DLBCL). Globally, clinical laboratories employ pragmatic but less than ideal immunohistochemical (IHC) assay for COO classification. Novel RNA-based platforms using routine pathology samples are emerging as new gold standard and offer unique opportunities for assay standardisation for laboratories across the world. We evaluated our IHC protocols against RNA-based technologies to determine concordance; additionally, we gauged the impact of preanalytical variation on the performance of Lymph2Cx assay.

Methods: Diagnostic biopsies (n=104) were examined for COO classification, employing automated RNA digital quantification assay (Lymph2Cx). Results were equated against IHC-based COO categorisation. Assay performance was assessed through its impact on overall survival (OS).

Results: 96 (92%) informative samples were labelled as GCB (38/96; 40%) and non-GCB (58/96; 60%) by IHC evaluation. Lymph2Cx catalogued 36/96 (37%) samples as GCB, 45/96 (47%) as ABC and 15/96 (16%) as unclassified. Lymph2Cx being reference, IHC protocol revealed sensitivity of 81% for ABC and 75% for GCB categorisation and positive predictive value of 81% versus 82%, respectively. Lymph2Cx-based COO classification performed superior to Hans algorithm in predicting OS (log rank test, p=0.017 vs p=0.212).

Conclusions: Our report show that current IHC-based protocols for COO classification of DLBCL at UKM Malaysia are in line with previously reported results and marked variation in preanalytical factors do not critically impact Lymph2Cx assay quality.
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http://dx.doi.org/10.1136/jclinpath-2017-204548DOI Listing
March 2018

Acute Myeloid Leukemia (AML): Upregulation of BAALC/MN1/MLLT11/EVI1 Gene Cluster Relate With Poor Overall Survival and a Possible Linkage With Coexpression of MYC/BCL2 Proteins.

Appl Immunohistochem Mol Morphol 2018 08;26(7):483-488

Departments of Pathology and Laboratory Medicine, Division of Hematology and Transfusion Medicine, Calgary Laboratory Services, Foothills Medical Centre.

Background: Molecular heterogeneity accounts for the variable and often poor prognosis in acute myeloid leukemia (AML). The current risk stratification strategy in clinical practice is limited to karyotyping and limited molecular studies screening for genetic mutations such as FLT-3 and NPM1. There is opportunity to identify further molecular prognostic markers, which may also lay the groundwork for the development of novel targeted therapies. Complex molecular technologies require transition into widely available laboratory platforms, for better integration into routine clinical practice.

Method: In a defined subset (MYC/BCL2 or MYC/BCL2) of AML patients (n=20), we examined expression signature of several genes (n=12) of established prognostic value in AML. RNA expression and MYC/BCL2 protein pattern was correlated with 3 cytogenetic risk groups and overall survival.

Results: K-means++ unsupervised clustering defined 2 distinct groups with high and low transcript levels of BAALC/MN1/MLLT11/EVI1/SOCS2 genes (>2.5-fold difference; P<0.001). This mRNA signature trended with higher prevalence of MYC/BCL2 coexpression (P<0.057) and poor overall survival (P<0.036), but did not correlate with conventional cytogenetic risk groups (P<0.084).

Conclusions: This pilot study provides useful data, which may help further refine the prognostic scheme of AML patients outside conventional cytogenetic risk groups. It also presents some biological rationale for future studies to explore the use of novel agents targeting MYC and/or BCL2 genes in combination with conventional chemotherapy protocols for AML.
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http://dx.doi.org/10.1097/PAI.0000000000000452DOI Listing
August 2018

MS4A4A: a novel cell surface marker for M2 macrophages and plasma cells.

Immunol Cell Biol 2017 08 17;95(7):611-619. Epub 2017 Mar 17.

Department of Biochemistry and Molecular Biology, and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.

MS4A4A is a member of the membrane-spanning, four domain family, subfamily A (MS4A) that includes CD20 (MS4A1), FcRβ (MS4A2) and Htm4 (MS4A3). Like the first three members of this family, transcription of MS4A4A appears to be limited to hematopoietic cells. To evaluate expression of the MS4A4A protein in hematopoietic cell lineages and subsets we generated monoclonal antibodies against extracellular epitopes for use in flow cytometry. In human peripheral blood we found that MS4A4A is expressed at the plasma membrane in monocytes but not in granulocytes or lymphocytes. In vitro differentiation of monocytes demonstrated that MS4A4A is expressed in immature but not activated dendritic cells, and in macrophages generated in the presence of interleukin-4 ('alternatively activated' or M2 macrophages) but not by interferon-γ and lipopolysaccharide ('classically' activated or M1 macrophages). MS4A4A was expressed in the U937 monocytic cell line only after differentiation. In normal bone marrow, MS4A4A was expressed in mature monocytes but was undetected, or detected at only a low level, in myeloid/monocytic precursors, as well as their malignant counterparts in patients with various subtypes of myeloid leukemia. Although MS4A4A was not expressed in healthy B lymphocytes, it was highly expressed in normal plasma cells, CD138+ cells from multiple myeloma patients, and bone marrow B cells from a patient with mantle cell lymphoma. These findings suggest immunotherapeutic potential for MS4A4A antibodies in targeting alternatively activated macrophages such as tumor-associated macrophages, and in the treatment of multiple myeloma and mantle cell lymphoma.
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http://dx.doi.org/10.1038/icb.2017.18DOI Listing
August 2017

Multiplexed automated digital quantification of fusion transcripts: comparative study with fluorescent in-situ hybridization (FISH) technique in acute leukemia patients.

Diagn Pathol 2016 Sep 15;11(1):89. Epub 2016 Sep 15.

Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada.

Background: The World Health Organization (WHO) classification system defines recurrent chromosomal translocations as the sole diagnostic and prognostic criteria for acute leukemia (AL). These fusion transcripts are pivotal in the pathogenesis of AL. Clinical laboratories universally employ conventional karyotype/FISH to detect these chromosomal translocations, which is complex, labour intensive and lacks multiplexing capacity. Hence, it is imperative to explore and evaluate some newer automated, cost-efficient multiplexed technologies to accommodate the expanding genetic landscape in AL.

Methods: "nCounter® Leukemia fusion gene expression assay" by NanoString was employed to detect various fusion transcripts in a large set samples (n = 94) utilizing RNA from formalin fixed paraffin embedded (FFPE) diagnostic bone marrow biopsy specimens. This series included AL patients with various recurrent translocations (n = 49), normal karyotype (n = 19), or complex karyotype (n = 21), as well as normal bone marrow samples (n = 5). Fusion gene expression data were compared with results obtained by conventional karyotype and FISH technology to determine sensitivity/specificity, as well as positive /negative predictive values.

Results: Junction probes for PML/RARA; RUNX1-RUNX1T1; BCR/ABL1 showed 100 % sensitivity/specificity. A high degree of correlation was noted for MLL/AF4 (85 sensitivity/100 specificity) and TCF3-PBX1 (75 % sensitivity/100 % specificity) probes. CBFB-MYH11 fusion probes showed moderate sensitivity (57 %) but high specificity (100 %). ETV6/RUNX1 displayed discordance between fusion transcript assay and FISH results as well as rare non-specific binding in AL samples with normal or complex cytogenetics.

Conclusions: Our study presents preliminary data with high correlation between fusion transcript detection by a throughput automated multiplexed platform, compared to conventional karyotype/FISH technique for detection of chromosomal translocations in AL patients. Our preliminary observations, mandates further vast validation studies to explore automated molecular platforms in diagnostic pathology.
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http://dx.doi.org/10.1186/s13000-016-0541-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024455PMC
September 2016

Acute myeloid leukaemia: expression of MYC protein and its association with cytogenetic risk profile and overall survival.

Hematol Oncol 2017 Sep 8;35(3):350-356. Epub 2016 Feb 8.

Department of Pathology & Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, Alberta, Canada.

Acute myeloid leukaemia (AML) is a clinically aggressive disease with marked genetic heterogeneity. Cytogenetic abnormalities provide the basis for risk stratification into clinically favourable, intermediate, and unfavourable groups. There are additional genetic mutations, which further influence the prognosis of patients with AML. Most of these result in molecular aberrations whose downstream target is MYC. It is therefore logical to study the relationship between MYC protein expression and cytogenetic risk groups. We studied MYC expression by immunohistochemistry in a large cohort (n = 199) of AML patients and correlated these results with cytogenetic risk profile and overall survival (OS). We illustrated differential expression of MYC protein across various cytogenetic risk groups (p = 0.03). Highest expression of MYC was noted in AML patients with favourable cytogenetic risk group. In univariate analysis, MYC expression showed significant negative influence of OS in favourable and intermediate cytogenetic risk group (p = 0.001). Interestingly, MYC expression had a protective effect in the unfavourable cytogenetic risk group. In multivariate analysis, while age and cytogenetic risk group were significant factors influencing survival, MYC expression by immunohistochemistry methods also showed some marginal impact (p = 0.069). In conclusion, we have identified differential expression of MYC protein in relation to cytogenetic risk groups in AML patients and documented its possible impact on OS in favourable and intermediate cytogenetic risk groups. These preliminary observations mandate additional studies to further investigate the routine clinical use of MYC protein expression in AML risk stratification. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/hon.2279DOI Listing
September 2017

Low Counts of B Cells, Natural Killer Cells, Monocytes, Dendritic Cells, Basophils, and Eosinophils are Associated with Postengraftment Infections after Allogeneic Hematopoietic Cell Transplantation.

Biol Blood Marrow Transplant 2016 Jan 9;22(1):37-46. Epub 2015 Sep 9.

Department of Medicine, University of Calgary, Calgary, Alberta, Canada.

Hematopoietic cell transplant (HCT) recipients are immunocompromised and thus predisposed to infections. We set out to determine the deficiency of which immune cell subset(s) may predispose to postengraftment infections. We determined day 28, 56, 84, and 180 blood counts of multiple immune cell subsets in 219 allogeneic transplant recipients conditioned with busulfan, fludarabine, and Thymoglobulin. Deficiency of a subset was considered to be associated with infections if the low subset count was significantly associated with subsequent high infection rate per multivariate analysis in both discovery and validation cohorts. Low counts of monocytes (total and inflammatory) and basophils, and low IgA levels were associated with viral infections. Low plasmacytoid dendritic cell (PDC) counts were associated with bacterial infections. Low inflammatory monocyte counts were associated with fungal infections. Low counts of total and naive B cells, total and CD56(high) natural killer (NK) cells, total and inflammatory monocytes, myeloid dendritic cells (MDCs), PDCs, basophils and eosinophils, and low levels of IgA were associated with any infections (due to any pathogen or presumed). In conclusion, deficiencies of B cells, NK cells, monocytes, MDCs, PDCs, basophils, eosinophils, and/or IgA plasma cells appear to predispose to postengraftment infections.
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http://dx.doi.org/10.1016/j.bbmt.2015.09.003DOI Listing
January 2016

Concomitant high expression of Toll-like receptor (TLR) and B-cell receptor (BCR) signalling molecules has clinical implications in mantle cell lymphoma.

Hematol Oncol 2017 Mar 10;35(1):79-86. Epub 2015 Sep 10.

Division of Hematology and Transfusion Medicine, Department of Pathology and Laboratory Medicine, University of Calgary/Calgary Laboratory Services (CLS), Calgary, Alberta, Canada.

Mantle cell lymphoma (MCL) is an aggressive disease with frequent relapse. Targeted therapies against B-cell receptor (BCR) molecules have demonstrated improved outcomes in relapsed cases. However, clinical responses are slow and selective, with failure to attain complete remission in a significant subset of patients. Complex interaction of BCR signal transduction with toll-like receptor (TLR) and other pathways in MCL remains unknown, thus averting progress in development of targeted therapies. We have performed detailed digital quantification of BCR/TLR signalling molecules and their effector pathways in a cohort (n = 81) of MCL patients and correlated these data with overall survival. Hierarchical clustering model based on BCR/TLR genes revealed two distinct (BCR and BCR ) subsets of patients (n = 32; 40%) with significant differences in expression (>1.5-fold change; p < 0.05). Higher levels of BTK/SYK/BLNK/CARD11/PLCG signalosome and lower expression of MALT1/BCL10 genes suggested tonic pattern of BCR activation. Amplified expression of TLR6/TLR7/TLR9 was noted in concert with hyper-responsiveness of BCR machinery. MYD88, a key TLR adaptor molecule, was not upregulated in any of these clusters, which may suggest a 'cross-talk' between BCR and TLR pathways. In sync with BCR/TLR signalling, we recorded significantly enhanced expression of genes associated with NF-kB pathway in BCR subset of MCL patients. On univariate analysis, the BCR patients showed a trend towards inferior clinical response to a standardized treatment protocol, compared with the BCR group (log rank, p = 0.043). In conclusion, we have identified hyperactive BCR/TLR signalling pathways and their effector downstream targets in a subset of MCL patients and associated it with poor clinical outcomes. Our study provides quantitative evidence at RNA expression level of possible concomitant collaboration between TLR and BCR signalling molecules in MCL. These data will provide further insights for future functional studies and, hence, development of targeted therapies for MCL patients. Copyright © 2015 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/hon.2251DOI Listing
March 2017

CD10-positive mantle cell lymphoma: biologically distinct entity or an aberrant immunophenotype? Insight, through gene expression profile in a unique case series.

J Clin Pathol 2015 Oct 29;68(10):844-8. Epub 2015 Jun 29.

Division of Hematology and Transfusion Medicine, Department of Pathology and Laboratory Medicine, University of Calgary/Calgary Laboratory Services (CLS), Calgary, Alberta, Canada.

Background: Mantle cell lymphoma (MCL) is an aggressive disease with genetic heterogeneity and discrete clinical subtypes. MCL is rarely CD10 positive. These cases raise the question whether a subset of MCL may be germinal centre (GC) derived, and have distinct clinicopathological characteristics.

Aims And Methods: A series of nine CD10-positive MCL cases is described herein. The clinicopathological and immunophenotypic features, immunoglobulin somatic hypermutation (SHM) status and gene expression profile (GEP) data are detailed. These features were compared with two independent sets (n=20, each) of CD10-negative MCL cases (controls), which were randomly selected from our institutional registry.

Results: GEP showed distinct expression of a GC signature in CD10-positive MCL cases with minimal impact on downstream signalling pathways. There were no significant differences in the clinicopathological features or clinical outcome between our CD10-positive and CD10-negative MCL cases. The frequency of SHM was comparable with established data.

Conclusions: This study provides convincing evidence that CD10 expression is related to a distinct GC signature in MCL cases, but without clinical or biological implications.
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http://dx.doi.org/10.1136/jclinpath-2015-202955DOI Listing
October 2015

Performance of the CellaVision(®) DM96 system for detecting red blood cell morphologic abnormalities.

J Pathol Inform 2015 24;6:11. Epub 2015 Feb 24.

Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, AB T2L 2K8, Canada ; Calgary Laboratory Services, Calgary, AB T2L 2K8, Canada.

Background: Red blood cell (RBC) analysis is a key feature in the evaluation of hematological disorders. The gold standard light microscopy technique has high sensitivity, but is a relativity time-consuming and labor intensive procedure. This study tested the sensitivity and specificity of gold standard light microscopy manual differential to the CellaVision(®) DM96 (CCS; CellaVision, Lund, Sweden) automated image analysis system, which takes digital images of samples at high magnification and compares these images with an artificial neural network based on a database of cells and preclassified according to RBC morphology.

Methods: In this study, 212 abnormal peripheral blood smears within the Calgary Laboratory Services network of hospital laboratories were selected and assessed for 15 different RBC morphologic abnormalities by manual microscopy. The same samples were reassessed as a manual addition from the instrument screen using the CellaVision(®) DM96 system with 8 microscope high power fields (×100 objective and a 22 mm ocular). The results of the investigation were then used to calculate the sensitivity and specificity of the CellaVision(®) DM96 system in reference to light microscopy.

Results: The sensitivity ranged from a low of 33% (RBC agglutination) to a high of 100% (sickle cells, stomatocytes). The remainder of the RBC abnormalities tested somewhere between these two extremes. The specificity ranged from 84% (schistocytes) to 99.5% (sickle cells, stomatocytes).

Conclusions: Our results showed generally high specificities but variable sensitivities for RBC morphologic abnormalities.
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http://dx.doi.org/10.4103/2153-3539.151922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355826PMC
March 2015

De Novo Acute Myeloid Leukemia in Adults: Suppression of MicroRNA-223 is Independent of LMO2 Protein Expression BUT Associate With Adverse Cytogenetic Profile and Undifferentiated Blast Morphology.

Appl Immunohistochem Mol Morphol 2015 Nov-Dec;23(10):733-9

*Calgary Laboratory Services (CLS), Department of Pathology & Laboratory Medicine, Division of Hematology & TM ‡Calgary Laboratory Services (CLS), Department of Pathology & Laboratory Medicine, Division of Anatomic Pathology, University of Calgary, Calgary, AB, Canada †Department of Pathology and Laboratory Medicine (GE) and Oncology (AMA), Prince Sultan Military Medical City, Riyadh, Saudi Arabia.

MicroRNA (MIR) signatures are critical to pathobiology and prognosis of acute myeloid leukemia (AML). MIR223 is expressed at low levels in progenitor cells, whereas high expression is induced by granulocytic differentiation. Novel-targeted therapies through epigenetic manipulation of MIR223 regulators are being explored in AML but correlative data between established clinical prognostic markers and MIR223 expression in AML is lacking. MIR223 has inverse relationship with LMO2 protein expression and our group has recently reported a close association between LMO2 protein expression and chromosomal findings in AML patients. In this study, we examined the expression of MIR223 in a large cohort of AML patients and correlated it with LMO2 protein expression, cytogenetic data, degree of differentiation [French-American and British (FAB)/World Health Organization classifications], and overall survival. MIR223 expression was upregulated in only a subset of patients (37%). Suppression of MIR223 was more frequent among patients with aneuploid karyotype compared with diploid karyotype (P=0.005). In AML, not otherwise specified category, AML with maturation (FAB-M2) showed higher levels of MIR223 when compared with either AML without maturation (FAB M0/M1) (P=0.001); AML with monoblastic differentiation (FAB M4/M5) (P=0.004) or AML with myelodysplasia-related changes (P=0.011). Among cytogenetic risk groups, suppression of MIR223 was universal (>95%) in high-risk group when compared with intermediate-risk group (P=0.004). No correlation between MIR223 and LMO2 protein expression was identified. In conclusion, we have shown that suppression of MIR223 expression, as compared with controls, is associated with lack of differentiation and adverse cytogenetic profile, but unrelated with LMO2 protein expression or overall survival.
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http://dx.doi.org/10.1097/PAI.0000000000000145DOI Listing
August 2016

Differential expression of Toll-like receptor (TLR) and B cell receptor (BCR) signaling molecules in primary diffuse large B-cell lymphoma of the central nervous system.

J Neurooncol 2015 Jan 13;121(2):289-96. Epub 2014 Nov 13.

Division of Hematology and Transfusion Medicine, Department of Pathology and Laboratory Medicine, University of Calgary/Calgary Laboratory Services (CLS), Calgary, AB, Canada.

Primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL) is a distinct and aggressive lymphoma that is confined to CNS. Since, central nervous system is barrier-protected and immunologically silent; role of TLR/BCR signaling in pathogenesis and biology of CNS DLBCL is intriguing. Genomic mutations in key regulators of TLR/BCR signaling pathway (MYD88/CD79B/CARD11) have recently been reported in this disease. These observations raised possible implications in novel targeted therapies; however, expression pattern of molecules related to TLR/BCR pathways in this lymphoma remains unknown. We have analyzed the expression of 19 genes encoding TLR/BCR pathways and targets in CNS DLBCLs (n = 20) by Nanostring nCounter™ analysis and compared it with expression patterns in purified reactive B-lymphocytes and systemic diffuse large B cell lymphoma (DLBCL) (n = 20). Relative expression of TLR4, TLR5, TLR9, CD79B and BLNK was higher in CNS DLBCLs than in control B-lymphocytes; where as TLR7, MALT1, BCL10, CD79A and LYN was lower in CNS DLBCLs (P < 0.0001). When compared with systemic DLBCL samples, higher expression of TLR9, CD79B, CARD11, LYN and BLNK was noted in CNS DLBCL (>1.5 fold change; P < 0.01). The B cell receptor molecules like BLNK and CD79B were also associated with higher expression of MYD88 dependent TLRs (TLR4/5/9). In conclusion, we have shown over expression of TLR/BCR related genes or their targets, where genomic mutations have commonly been identified in CNS DLBCL. We have also demonstrated that TLR over expression closely relate with up regulation of genes associated with BCR pathway like CD79B/BLNK and CARD11, which play an important role in NF-kB pathway activation. Our results provide an important insight into the possibility of TLR and/or B-cell receptor signaling molecules as possible therapeutic targets in CNS DLBCL.
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http://dx.doi.org/10.1007/s11060-014-1655-3DOI Listing
January 2015

Protein Expression for Novel Prognostic Markers (Cyclins D1, D2, D3, B1, B2, ITGβ7, FGFR3, PAX5) Correlate With Previously Reported Gene Expression Profile Patterns in Plasma Cell Myeloma.

Appl Immunohistochem Mol Morphol 2015 May-Jun;23(5):327-33

Department of Pathology & Laboratory Medicine, Division of Hematology and Transfusion Medicine, University of Calgary/Calgary Laboratory Services (CLS), Calgary, AB, Canada.

Among plasma cell myeloma (PCM) patients, gene expression profiling (GEP)-based molecular classification has proven to be an independent predictor of survival, after autologous stem cell transplantation. However, GEP has limited routine clinical applicability given its complex methodology, high cost, and limited availability in clinical laboratories. In this study, we have evaluated biomarkers identified from GEP discoveries, utilizing immunohistochemistry (IHC) platform in a cohort of PCM patients. IHC staining for cyclins B1, B2, D1, D2, D3, FGFR3, PAX5, and integrin β7 (ITGβ7) was performed on the bone marrow biopsies of 93 newly diagnosed PCM patients. Expression of FGFR3 was noted in 10 (11%) samples correlating completely with t(4;14)(p16;q32) results (P<0.001); however, the association between FGFR3 and cyclin D2 expression was not significant (P=0.14). ITGβ7 expression was present in 9/93 (9%) patients and all these samples also demonstrated upregulated expression of cyclin D2 (P=0.014). Expression of cyclins D1, D2, and D3 was variable in this cohort. Positive protein expression of cyclin D1 was noted in 30/93 (32%), D2 in 17/93 (18%), and D3 in 5/93 (5%) samples. Coexpression of cyclins D1 and D2 was observed in 13/93 (14%) samples, whereas 28 (30%) samples were negative for all the 3 cyclin D proteins. Cyclin B1 was not expressed in any sample, despite adequate staining in positive controls. Cyclin B2 was expressed in 33/93 (35%) and PAX5 protein was noted in 7/93 (8%) samples. In summary, we have demonstrated that mRNA-based prognostic markers can be detected by routine IHC in decalcified bone marrow samples. This approach may provide a useful tool for the wider adoption of prognostic makers for risk stratification of PCM patients. We anticipate that such an approach might allow patients with high-risk immunoprofiles to be considered for other potential novel therapeutic agents, potentially sparing some patients the toxicity of stem cell transplant.
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http://dx.doi.org/10.1097/PAI.0000000000000089DOI Listing
February 2016

PARP1 expression in mantle cell lymphoma: the utility of PARP1 immunohistochemistry and its relationship with markers of DNA damage.

Hematol Oncol 2015 Dec 20;33(4):159-65. Epub 2014 Aug 20.

Department of Pathology & Laboratory Medicine, University of Calgary/Calgary Laboratory Services, Calgary, AB, Canada.

Mantle cell lymphoma (MCL) is an aggressive disease with poor overall survival, attributable in part to frequent defects of the DNA repair genes. In such malignancies, additional inhibition of the ubiquitous DNA damage repair protein, poly-ADP ribose polymerase-1 (PARP1) has shown enhanced cytotoxicity (so-called synthetic lethality). We studied PARP1 expression in a series of clinical cases of MCL, with the secondary aim to ascertain the relationship between PARP1 expression and DNA repair gene expression (namely ATM and p53) by immunohistochemical methods. We also examined the relationship between PARP1 expression and the well-established prognostic biomarker Ki-67, in addition to correlating PARP1 expression with the overall survival. From amongst our series of 79 unselected cases of MCL, we detected PARP1 expression in all but two cases with variable intensity. We also noted correlations between PARP1 expression and ATM and p53 expression. As described in previous studies, we identified a significant survival difference on the basis of Ki-67 and p53 expression. When digital H-score analysis of PARP1 expression was performed, there was a distinct survival advantage noted in patients with lower levels of expression. When our biomarker data were assessed by Cox regression, furthermore, the dominant effects of p53 and PARP1 expression were highlighted. Our data support the need for further research into the potential utility of PARP1 as a biomarker in MCL and for the potential direction of future PARP1 inhibitor-targeted therapy studies.
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http://dx.doi.org/10.1002/hon.2160DOI Listing
December 2015

Accuracy of the CellaVision DM96 platform for reticulocyte counting.

J Pathol Inform 2014 26;5(1):17. Epub 2014 May 26.

Department of Pathology and Laboratory Medicine, Division of Hematology and Transfusion Medicine, Calgary Laboratory Services, University of Calgary, Calgary, Alberta, Canada.

Context: Many hematology laboratories have adopted semi-automated digital platforms for routine use and the evidence supporting their use is increasing.

Aims: The CellaVision platforms are among the most thoroughly studied digital hematology platforms; we wished to determine the accuracy of CellaVision for reticulocyte counting.

Design Materials And Methods: We compared reticulocyte counts performed manually, using the Beckman Coulter LH750 automated analyzer and with the CellaVision DM96 platform. We analyzed the results for pair-wise correlation and bias, and precision.

Statistical Analyses Used: Analyses were performed using Statistical Package for the Social Sciences software (SPSS), including Spearman's rho correlation coefficient, Friedman's two-way Analysis Of Variance (ANOVA) for comparison of distributions; bias was compared by way of mean and standard deviation.

Results: The CellaVision reticulocyte counts correlated most strongly with those of the analyzer (often considered the benchmark test); the reticulocyte count distributions were noted not to be significantly different from each other across all three methods. The mean and standard deviation of bias were lowest in the comparison of CellaVision and LH750 counts.

Conclusions: Our data provide additional support for the accuracy of digital hematology applications using the CellaVision DM96 platform.
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http://dx.doi.org/10.4103/2153-3539.133127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060401PMC
July 2014

B-lymphoblastic leukemia/lymphoma: overexpression of nuclear DNA repair protein PARP-1 correlates with antiapoptotic protein Bcl-2 and complex chromosomal abnormalities.

Hum Pathol 2014 Aug 18;45(8):1582-7. Epub 2014 Apr 18.

Department of Pathology & Laboratory Medicine, University of Calgary and Calgary Laboratory Services (CLS), Calgary, Alberta, Canada T2N 2T9. Electronic address:

Poly(ADP-ribose) polymerase-1 (PARP-1) and Bcl-2 are emerging as therapeutic targets in various cancers. The former is a DNA repair protein associated with genomic stability and apoptosis, whereas the latter is an antiapoptotic protein having a DNA repair function through inhibition of PARP-1. Because genomic stability is critical for prognosis in B-lymphoblastic leukemia/lymphoma (B-ALL), we studied the expression of PARP-1 and Bcl-2 proteins in patients with B-ALL of different ages and compared the results with cytogenetic data. The PARP-1 protein was overexpressed in about two-thirds (61%) of patients with B-ALL. It had a nuclear location, whereas Bcl-2 protein was cytosolic. Expression of the 2 proteins showed a highly positive correlation (ρ = 0.367; P < .001). Overexpression of PARP-1 correlated with a complex karyotype (P = .030), and this correlation remained significant for coexpression of PARP-1 and Bcl-2 proteins (χ(2) = 7.498; P = .024) as well as after exclusion of pediatric patients (n = 9, P = .042). Overexpression of PARP-1 was not significantly more common in diploid versus aneuploid karyotypes (50% versus 59%, P = .610). The PARP-1 protein showed no correlation with specific chromosomal abnormalities associated with prognosis in B-ALL, as defined by the World Health Organization. In conclusion, high expression of the PARP-1 protein among patients with B-ALL is related to a complex karyotype and Bcl-2 positivity. Although these findings require validation in a larger population, the observations will be valuable in planning therapeutic trials (such as of PARP inhibitors and BH3 mimetics).
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http://dx.doi.org/10.1016/j.humpath.2013.11.024DOI Listing
August 2014

Immune cell subset counts associated with graft-versus-host disease.

Biol Blood Marrow Transplant 2014 Apr 6;20(4):450-62. Epub 2014 Jan 6.

Department of Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.

Graft-versus-host disease (GVHD) is a major transplantation complication. The purpose of this study was to measure immune cell subsets by flow cytometry early after transplantation (before median day of GVHD onset) to identify subsets that may play a role in GVHD pathogenesis. We also measured the subsets later after transplantation to determine which subsets may be influenced by GVHD or its treatment. We studied 219 patients. We found that acute GVHD (aGVHD) was preceded by high counts of CD4 T cells and CD8 T cells. It was followed by low counts of total and naive B cells, total and cytolytic NK cells, and myeloid and plasmacytoid dendritic cells. Chronic GVHD (cGVHD) was preceded by low counts of memory B cells. In conclusion, both CD4 and CD8 T cells appear to play a role in the pathogenesis of aGVHD. Generation of B cells, NK cells, and dendritic cells may be hampered by aGVHD and/or its treatment. Memory B cells may inhibit the development of cGVHD.
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http://dx.doi.org/10.1016/j.bbmt.2014.01.002DOI Listing
April 2014

LMO2 (LIM domain only 2) is expressed in a subset of acute myeloid leukaemia and correlates with normal karyotype.

Histopathology 2014 Jan 6;64(2):226-33. Epub 2013 Nov 6.

Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada.

Aim: LMO2 is a transcription factor that plays a key role in haematopoiesis. Expression of LMO2 has been demonstrated in germinal centre B cells, various B cell lymphomas and T lymphoblastic lymphoma/leukaemia (T-ALL), but has not been studied extensively in acute myeloid leukaemia (AML).

Methods: We studied LMO2 expression by immunohistochemistry in biopsies from a cohort of AML patients (n = 196) and correlated it with established prognostic factors such as age, bone marrow morphology and cytogenetic findings.

Results: Forty per cent (79 of 196) of the samples from AML patients showed moderate/strong expression of LMO2 protein. LMO2 expression showed a significant positive correlation with normal cytogenetics (65% versus 24%, P < 0.0001) and a moderately negative correlation with complex karyotype [rs (98) = -0.218, P < 0.002]. AML associated with core binding factor [(t(8;21)/inv(16)/t(16;16)] had low LMO2 expression compared to diploid karyotype (29% versus 65%; P = 0.013). Expression of LMO2 protein exhibited an insignificant association with age (P = 0.197). Lower expression of LMO2 protein was noted in AML associated with myelodysplasia-related changes, compared to AML subtypes based on FAB classification (M0-M7) (21% versus 44%, P = 0.0187).

Conclusions: LMO2 is expressed in a subset of AML patients and is associated with normal karyotype, which is different from T-ALL, where specific translocation (11p13) mediates protein expression.
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http://dx.doi.org/10.1111/his.12242DOI Listing
January 2014

Results of a prospective phase II trial evaluating interim positron emission tomography-guided high dose therapy for poor prognosis diffuse large B-cell lymphoma.

Leuk Lymphoma 2014 Sep 17;55(9):2064-70. Epub 2014 Feb 17.

Medicine and Oncology, University of Calgary , Calgary , Canada.

Patients with diffuse large B-cell lymphoma (DLBCL) with a poor prognosis based upon advanced stage and elevated serum lactate dehydrogenase achieve a 3-4-year progression-free survival (PFS) of only 55%. The role of interim fluoro-2-deoxy-d-glucose (FDG) positron emission tomography (PET) to guide use of upfront high dose chemotherapy (HDCT) and autologous stem cell transplant (ASCT) for patients with poor prognosis DLBCL is unproven. A prospective phase II clinical trial was designed to evaluate the outcomes of HDCT/ASCT for patients with poor prognosis DLBCL aged 18-65 years who had unfavorable interim restaging PET scans. Of the 70 eligible patients, 36 had unfavorable and 34 favorable interim PET responses, with 3-year PFS rates of 65.2% and 52.7%, respectively. In conclusion, favorable interim PET response as defined in this study is not associated with improved PFS rates for patients with poor prognosis DLBCL treated with RCHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). A phase III trial evaluating this PET-guided approach is not warranted.
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http://dx.doi.org/10.3109/10428194.2013.862242DOI Listing
September 2014

Expression of MS4A and TMEM176 Genes in Human B Lymphocytes.

Front Immunol 2013 15;4:195. Epub 2013 Jul 15.

Department of Biochemistry and Molecular Biology, Snyder Institute for Chronic Diseases, University of Calgary , Calgary, AB , Canada.

The MS4A gene family in humans includes CD20 and at least 15 other genes. CD20 exists as homo-oligomers in the plasma membrane, however different MS4A proteins expressed in the same cell may hetero-oligomerize. Given the importance of CD20 in B-cell function and as a therapeutic target, we sought to explore the potential for CD20 hetero-oligomerization with other MS4A proteins. We investigated expression in primary human B-cells of the four MS4A genes previously shown to be expressed in human B-cell lines (MS4A4A, MS4A6A, MS4A7, MS4A8B), as well as two genes comprising the closely related TMEM176 gene family, with a view to identifying candidates for future investigation at the protein level. TMEM176A and TMEM176B transcripts were either not detected, or were detected at relatively low levels in a minority of donor B-cell samples. MS4A4A and MS4A8B transcripts were not detected in any normal B-cell sample. MS4A6A and MS4A7 transcripts were detected at low levels in most samples, however the corresponding proteins were not at the plasma membrane when expressed as GFP conjugates in BJAB cells. We also examined expression of these genes in chronic lymphocytic leukemia (CLL), and found that it was similar to normal B-cells with two exceptions. First, whereas MS4A4A expression was undetected in normal B-cells, it was expressed in 1/14 CLL samples. Second, compared to expression levels in normal B-cells, MS4A6A transcripts were elevated in 4/14 CLL samples. In summary, none of the MS4A/TMEM176 genes tested was expressed at high levels in normal or in most CLL B-cells. MS4A6A and MS4A7 were expressed at low levels in most B-cell samples, however the corresponding proteins may not be positioned at the plasma membrane. Altogether, these data suggest that CD20 normally does not form hetero-oligomers with other MS4A proteins and that there are unlikely to be other MS4A proteins in CLL that might provide useful alternate therapeutic targets.
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http://dx.doi.org/10.3389/fimmu.2013.00195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711070PMC
July 2013

Platelet count estimation using the CellaVision DM96 system.

J Pathol Inform 2013 29;4:16. Epub 2013 Jun 29.

Calgary Laboratory Services, 9, 3535 Research Rd NW, Calgary, AB, T2L 2K8, Canada.

Introduction: Rapid and accurate determination of platelet count is an important factor in diagnostic medicine. Traditional microscopic methods are labor intensive with variable results and are highly dependent on the individual training. Recent developments in automated peripheral blood differentials using a computerized system have shown many advantages as a viable alternative. The purpose of this paper was to determine the reliability and accuracy of the CellaVision DM 96 system with regards to platelet counts.

Materials And Methods: One hundred twenty seven peripheral blood smears were analyzed for platelet count by manual microscopy, an automated hematology analyzer (Beckman Counter LH 780 or Unicel DXH 800 analyzers) and with the CellaVision DM96 system. Results were compared using the correlations and Bland-Altman plots.

Results: Platelet counts from the DM96 system showed an R(2) of 0.94 when compared to manual platelet estimates and an R(2) of 0.92 when compared to the automated hematology analyzer results. Bland-Altman plots did not show any systematic bias.
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http://dx.doi.org/10.4103/2153-3539.114207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709429PMC
July 2013

Performance of CellaVision DM96 in leukocyte classification.

J Pathol Inform 2013 29;4:14. Epub 2013 Jun 29.

Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, T2L 2K8, Canada.

Background: Leukocyte differentials are an important component of clinical care. Morphologic assessment of peripheral blood smears (PBS) may be required to accurately classify leukocytes. However, manual microscopy is labor intensive. The CellaVision DM96 is an automated system that acquires digital images of leukocytes on PBS, pre-classifies the cell type, and displays them on screen for a Technologist or Pathologist to approve or reclassify. Our study compares the results of the DM96 with manual microscopy.

Methods: Three hundred and fifty-nine PBS were selected and assessed by manual microscopy with a 200 leukocyte cell count. They were then reassessed using the CellaVision DM96 with a 115 leukocyte cell count including reclassification when necessary. Correlation between the manual microscopy results and the CellaVision DM96 results was calculated for each cell type.

Results: The correlation coefficients (r (2)) range from a high of 0.99 for blasts to a low of 0.72 for metamyelocytes.

Conclusions: The correlation between the CellaVision DM96 and manual microscopy was as good or better than the previously published data. The accuracy of leukocyte classification depended on the cell type, and in general, there was lower correlation for rare cell types. However, the correlation is similar to previous studies on the correlation of manual microscopy with an established reference result. Therefore, the CellaVision DM96 is appropriate for clinical implementation.
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http://dx.doi.org/10.4103/2153-3539.114205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709426PMC
July 2013

Reovirus modulates autophagy during oncolysis of multiple myeloma.

Autophagy 2013 Mar 15;9(3):413-4. Epub 2013 Jan 15.

Department of Oncology, University of Calgary, Calgary, Alberta, Canada.

Multiple myeloma (MM) is a clonal plasma cell malignancy that accounts for 10-15% of newly diagnosed hematological cancers. Although significant advances have been made in the treatment of MM the disease still remains incurable. The oncolytic potential of reovirus has previously been demonstrated by others and us and is currently in phase III clinical trials for solid tumors. In addition a phase I clinical trial has recently been initiated for MM. Despite the clinical activity, the mechanism(s) of cell death caused by reovirus in MM is yet not yet well elucidated. A comprehensive understanding of reovirus-mediated histology-specific cell death mechanisms is imperative if this therapeutic is to become a standard of care for patients. Previously we have shown that reovirus-mediated cell death of breast and prostate cancer is orchestrated via apoptosis. The present study demonstrates for the first time that in addition to inducing apoptosis reovirus also upregulates autophagy during oncolysis of MM.
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http://dx.doi.org/10.4161/auto.22867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590261PMC
March 2013

Immune reconstitution after anti-thymocyte globulin-conditioned hematopoietic cell transplantation.

Cytotherapy 2012 Nov 18;14(10):1258-75. Epub 2012 Sep 18.

University of Calgary/Alberta Health Services, Calgary, Alberta, Canada.

Background Aims: Anti-thymocyte globulin (ATG) is being used increasingly to prevent graft-versus-host disease (GvHD); however, its impact on immune reconstitution is relatively unknown. We (i) studied immune reconstitution after ATG-conditioned hematopoietic cell transplantation (HCT), (ii) determined the factors influencing the reconstitution, and (iii) compared it with non-ATG-conditioned HCT.

Methods: Immune cell subset counts were determined at 1-24 months post-transplant in 125 HCT recipients who received ATG during conditioning. Subset counts were also determined in 46 non-ATG-conditioned patients (similarly treated).

Results: (i) Reconstitution after ATG-conditioned HCT was fast for innate immune cells, intermediate for B cells and CD8 T cells, and very slow for CD4 T cells and invariant natural killer T (iNKT) (iNKT) cells. (ii) Faster reconstitution after ATG-conditioned HCT was associated with a higher number of cells of the same subset transferred with the graft in the case of memory B cells, naive CD4 T cells, naive CD8 T cells, iNKT cells and myeloid dendritic cells; lower recipient age in the case of naive CD4 T cells and naive CD8 T cells; cytomegalovirus recipient seropositivity in the case of memory/effector T cells; an absence of GvHD in the case of naive B cells; lower ATG serum levels in the case of most T-cell subsets, including iNKT cells; and higher ATG levels in the case of NK cells and B cells. (iii) Compared with non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT was slower for CD4 T cells, and faster for NK cells and B cells.

Conclusions: ATG worsens the reconstitution of CD4 T cells but improves the reconstitution of NK and B cells.
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http://dx.doi.org/10.3109/14653249.2012.715243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681879PMC
November 2012
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