Expression of "DNA damage response" pathway genes in diffuse large B-cell lymphoma: The potential for exploiting synthetic lethality.

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are two of the most prevalent non-Hodgkin's lymphoma subtypes. Despite advances, treatment resistance and patient relapse remain challenging issues. Our study aimed to scrutinize gene expression distinctions between DLBCL and FL, employing a cohort of 53 DLBCL and 104 FL samples that underwent rigorous screening for genetic anomalies. The NanoString nCounter assay evaluated 730 cancer-associated genes, focusing on densely tumorous areas in diagnostic samples. Employing the Lymph2Cx method, we determined the cell-of-origin (COO) for DLBCL cases. Our meticulous analysis, facilitated by Qlucore Omics Explorer software, unveiled a substantial 37% of genes with significantly differential expression patterns between DLBCL and FL, pointing to nuanced mechanistic disparities. Investigating the impact of FL disease stage and DLBCL COO on gene expression yielded minimal differences, prompting us to direct our attention to consistently divergent genes in DLBCL. Intriguingly, our Gene Set Enrichment Analysis spotlighted 21% of these divergent genes, converging on the DNA damage response (DDR) pathway, vital for cell survival and cancer evolution. Strong positive correlations among most DDR genes were noted, with key genes like BRCA1, FANCA, FEN1, PLOD1, PCNA, and RAD51 distinctly upregulated in DLBCL compared to FL and normal tissue controls. These findings were subsequently validated using RNA seq data on normal controls and DLBCL samples from public databases like The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases, enhancing the robustness of our results. Considering the established significance of these DDR genes in solid cancer therapies, our study underscores their potential applicability in DLBCL treatment strategies. In conclusion, our investigation highlights marked gene expression differences between DLBCL and FL, with particular emphasis on the essential DDR pathway. The identification of these DDR genes as potential therapeutic targets encourages further exploration of synthetic lethality-based approaches for managing DLBCL.

"Losing the Brakes"-Suppressed Inhibitors Triggering Uncontrolled Wnt/ß-Catenin Signaling May Provide a Potential Therapeutic Target in Elderly Acute Myeloid Leukemia.

Dysregulated Wnt/ß-catenin signal transduction is implicated in initiation, propagation, and poor prognosis in AML. Epigenetic inactivation is central to Wnt/ß-catenin hyperactivity, and Wnt/ß-catenin inhibitors are being investigated as targeted therapy. Dysregulated Wnt/ß-catenin signaling has also been linked to accelerated aging. Since AML is a disease of old age (>60 yrs), we hypothesized age-related differential activity of Wnt/ß-catenin signaling in AML patients. We probed Wnt/ß-catenin expression in a series of AML in the elderly (>60 yrs) and compared it to a cohort of pediatric AML (<18 yrs). RNA from diagnostic bone marrow biopsies (n = 101) were evaluated for key Wnt/ß-catenin molecule expression utilizing the NanoString platform. Differential expression of significance was defined as >2.5-fold difference (p < 0.01). A total of 36 pediatric AML (<18 yrs) and 36 elderly AML (>60 yrs) were identified in this cohort. Normal bone marrows (n = 10) were employed as controls. Wnt/ß-catenin target genes (MYC, MYB, and RUNX1) showed upregulation, while Wnt/ß-catenin inhibitors (CXXR, DKK1-4, SFRP1-4, SOST, and WIFI) were suppressed in elderly AML compared to pediatric AML and controls. Our data denote that suppressed inhibitor expression (through mutation or hypermethylation) is an additional contributing factor in Wnt/ß-catenin hyperactivity in elderly AML, thus supporting Wnt/ß-catenin inhibitors as potential targeted therapy.

Identification of Potential Therapeutic Targets for Plasmablastic Lymphoma Through Gene Expression Analysis: Insights into RAS and Wnt Signaling Pathways.

Plasmablastic lymphoma (PBL) is a rare and aggressive B-cell lymphoma with overlapping characteristics with diffuse large B-cell lymphoma (DLBCL) and multiple myeloma. Hyperactive Wnt signaling derails homeostasis and promotes oncogenesis and chemoresistance in DLBCL and multiple myeloma. Evidence suggests active cross-talk between the Wnt and RAS pathways impacting metastasis in solid cancers in which combined targeted therapies show effective results. Recent genomic studies in PBL demonstrated a high frequency of mutations linked with the RAS signaling pathway. However, the role of RAS and Wnt signaling pathway molecule expression in PBL remained unknown. We examined the expression of Wnt and RAS pathway-related genes in a well-curated cohort of PBL. Because activated B cells are considered immediate precursors of plasmablasts in B cell development, we compared this data with activated B-cell type DLBCL (ABC-DLBCL) patients, employing NanoString transcriptome analysis (770 genes). Hierarchical clustering revealed distinctive differential gene expression between PBL and ABC-DLBCL. Gene set enrichment analysis labeled the RAS signaling pathway as the most enriched (37 genes) in PBL, including upregulating critical genes, such as NRAS, RAF1, SHC1, and SOS1. Wnt pathway genes were also enriched (n = 22) by gene set enrichment analysis. Molecules linked with Wnt signaling activation, such as ligands or targets (FZD3, FZD7, c-MYC, WNT5A, WNT5B, and WNT10B), were elevated in PBL. Our data also showed that, unlike ABC-DLBCL, the deranged Wnt signaling activity in PBL was not linked with hyperactive nuclear factor κB and B-cell receptor signaling. In divergence, Wnt signaling inhibitors (CXXC4, SFRP2, and DKK1) also showed overexpression in PBL. The high expression of RAS signaling molecules reported may indicate linkage with gain-in-function RAS mutations. In addition, high expression of Wnt and RAS signaling molecules may pave pathways to explore benefiting from combined targeted therapies, as reported in solid cancer, to improve prognosis in PBL patients.

Primary testicular lymphoma demonstrates overexpression of the Wilms tumor 1 gene and different mRNA and miRNA expression profiles compared to nodal diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) shows a high degree of clinical and biological heterogeneity. Primary testicular lymphoma (PTL) is an extranodal variant of DLBCL associated with a higher risk of recurrence, including contralateral testicles and central nervous system sanctuary sites. Several molecular aberrations, including somatic mutation of MYD88, CD79B, and upregulation of NF-kB, PDL-1, and PDL-2, are thought to contribute to the pathogenesis and poor prognosis of PTL. However, additional biomarkers are needed that may improve the prognosis and help understand the PTL biology and lead to new therapeutic targets. RNA from diagnostic tissue biopsies of the PTL-ABC subtype and matched nodal DLBCL-ABC subtype patients was evaluated by mRNA and miRNA expression. Screening of 730 essential oncogenic genes was performed, and their epigenetic connections were examined using the nCounter PAN-cancer pathway, and Human miRNA assays with the nCounter System (NanoString Technologies). PTL and nodal DLBCL patients were comparable in age, gender, and putative cell of origin (p > 0.05). Wilms tumor 1 (WT1) expression in PTL exceeded that in nodal DLBCL (>6-fold; p = 0.01, FDR <0.01) and WT1 associated pathway genes THBS4, PTPN5, PLA2G2A, and IFNA17 were upregulated in PTL (>2.0-fold, p < 0.01, FDR <0.01). Additionally, miRNAs targeting WT1 (hsa15a-5p, hsa-miR-16-5p, has-miR-361-5p, has-miR-27b-3p, has-miR-199a-5p, has-miR-199b-5p, has-miR-132-3p, and hsa-miR-128-3p) showed higher expression in PTL compared to nodal DLBCL (≥2.0-fold; FDR 0.01). Lower expression of BMP7, LAMB3, GAS1, MMP7, and LAMC2 (>2.0-fold, p < 0.01) was observed in PTL compared to nodal DLBCL. This research revealed higher WT1 expression in PTL relative to nodal DLBCL, suggesting that a specific miRNA subset may target WT1 expression and impact the PI3k/Akt pathway in PTL. Further investigation is needed to explore WT1's biological role in PTL and its potential as a therapeutic target.

Donor Genetic Predisposition to High Interleukin-10 Production Appears Protective against Acute Graft-Versus-Host Disease.

The persistence of graft-versus-host disease (GVHD) as the principal complication of allogeneic hematopoietic cell transplantation (HCT) demonstrates that HLA matching alone is insufficient to prevent alloreactivity. We performed molecular and functional characterization of 22 candidate cytokine genes for their potential to improve matching in 315 myeloablative, 10/10 HLA-matched donor−recipient pairs. Recipients of a graft carrying the -1082GG IL10 gene promoter region variant had a three-fold lower incidence of grade II−IV acute GVHD compared to IL10-1082AA graft recipients (SHR = 0.25, p = 0.005). This was most evident in matched unrelated donor (MUD) transplants, where the greatest alloreactivity is expected. IL10-1082GG transplants did not experience an increased incidence of relapse, and, consequently, overall survival was two-fold higher in IL10-1082GG MUD transplants (HR = 0.17, p = 0.023). Longitudinal post-transplant measurements demonstrated that -1082GG is a high-IL10-producing and -expressing genotype with attenuated CD8+ T-cell reconstitution. High post-transplant donor chimerism in T- and myeloid-cells (>95%) confirmed a predominant donor, rather than recipient, genotype effect on immune function and aGVHD. To date, this is the first study to report corroborating genome-to-cellular evidence for a non-HLA donor immunogenetic variant that appears to be protective against GVHD. The incorporation of IL10 variants in donor selection criteria and clinical-management decisions has the potential to improve patient outcomes.

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

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.

Results of Octaplex for reversal of warfarin anticoagulation in patients with hip fracture.

BACKGROUND: Patients with hip fracture who present anticoagulated with warfarin often require reversal of anticoagulation for safe hip fracture surgery. Vitamin K is typically administered for this, but requires 24-48 hours for maximal effect. These patients have an increased delay to surgery and increased mortality. Octaplex is a prothrombin complex concentrate (PCC) that reverses warfarin anticoagulation in less than an hour. This study assesses the effectiveness and safety of Octaplex for reversal of warfarin anticoagulation for hip fracture surgery. METHODS: We reviewed the medical records of all patients with hip fracture in Calgary who received Octaplex between 2009 and 2015. Timing of admission, Octaplex administration and hip fracture surgery were recorded. Mortality and cardiac, thrombotic and orthopedic complications were assessed. RESULTS: Median time from Octaplex administration to an international normalized ratio of 1.4 or lower was 1.1 hours. The median time from admission to surgery was 22 hours. Thirty-day mortality was 15.2%, with 4 cases of cardiac arrest and 1 respiratory arrest. Patients who received both Octaplex and fresh frozen plasma (FFP) had a lower rate of 30-day survival than those who received only Octaplex (95.7% v. 60.0%, p = 0.002). CONCLUSION: There were significant rates of cardiac events and 30-day mortality among patients who received Octaplex, but this is unsurprising in this population with multiple medical comorbidities. We caution against administrering both FFP and a PCC in patients for warfarin reversal. Octaplex is effective for rapidly reversing warfarin anticoagulation and reducing delays to hip fracture surgery. Further study comparing Octaplex to reversal using only vitamin K is required.

CONTEXTE: Les patients avec fracture de la hanche qui sont sous anticoagulothérapie par warfarine au moment de consulter ont souvent besoin qu'on inverse leur anticoagulation pour être opérés sans danger. La vitamine K est généralement administrée à cette fin, mais il lui faut de 24 à 48 heures pour exercer son plein effet. Chez ces patients, le délai est plus long avant la chirurgie et la mortalité est plus élevée. Octaplex est un concentré de complexe prothrombique (CCP) qui inverse l'anticoagulation due à la warfarine en moins d'une heure. Cette étude évalue l'efficacité et l'innocuité d'Octaplex pour l'inversion de l'anticoagulation due à la warfarine lors d'une chirurgie pour fracture de la hanche. MÉTHODES: Nous avons passé en revue les dossiers médicaux de tous les patients avec fracture de la hanche à Calgary qui ont reçu Octaplex entre 2009 et 2015. Nous avons enregistré le moment de l'admission, de l'administration d'Octaplex et de la chirurgie pour fracture de la hanche. Nous avons évalué la mortalité et les complications cardiaques, thrombotiques et orthopédiques. RÉSULTATS: L'intervalle médian entre l'administration d'Octaplex et l'obtention d'un ratio international normalisé de 1,4 ou moins a été de 1,1 heure. L'intervalle médian entre l'admission et la chirurgie a été de 22 heures. La mortalité à 30 jours a été de 15,2 %, incluant 4 arrêts cardiaques et 1 arrêt respiratoire. Les patients qui ont reçu Octaplex et du plasma frais congelé (PFC) ont eu un taux de survie à 30 jours moins élevé que ceux qui ont reçu Octaplex seulement (95,7 % c. 60,0 %, p = 0,002). CONCLUSION: On a observé des taux significatifs d'événements cardiaques et de mortalité à 30 jours chez les patients traités par Octaplex, mais cela est peu surprenant dans cette population présentant plusieurs comorbidités médicales. Nous formulons une mise en garde contre l'utilisation de PFC et d'un CCP chez les patients soumis à une inversion de l'effet de la warfarine. Octaplex est efficace pour inverser rapidement l'anticoagulation due à la warfarine et accélérer l'accès à la chirurgie pour fracture de la hanche. Il faudra approfondir la recherche et comparer l'inversion par Octaplex plutôt que par la vitamine K seulement.

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

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.

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

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 (BCRhigh and BCRlow ) 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 BCRhigh subset of MCL patients. On univariate analysis, the BCRhigh patients showed a trend towards inferior clinical response to a standardized treatment protocol, compared with the BCRlow 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.

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

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.

SARA: a "new" low-frequency MNS antigen (MNS47) provides further evidence of the extreme diversity of the MNS blood group system.

BACKGROUND: Until recently, SARAH (SARA) was a low-frequency antigen within the 700 series (700.052). SARA was discovered in Australia and subsequently described in Canada where anti-SARA was implicated in severe hemolytic disease of the fetus and newborn (HDFN). This study investigated whether SARA could be recategorized into an existing, or novel, blood group system. STUDY DESIGN AND METHODS: Serologically typed Australian SARA family members (n = 9) were exome sequenced followed by bioinformatics analysis. Sanger sequencing of Exon 3 of GYPA of Australian (n = 9) and Canadian (n = 9) family members was then performed, as were peptide inhibition studies. RESULTS: Exome sequencing identified 499,329 single-nucleotide variants (SNVs) within the nine individuals. Filtering excluded SNVs with an NCBI dbSNP ID (n = 482,177) and non-protein coding SNVs (n = 14,008); for the remaining 3144 SNVs, only one, c.240G>T of GYPA encoding p.Arg80Ser, was present in all six SARA-positive individuals. Sanger sequencing confirmed the presence of c.240G>T in the Australian SARA-positive individuals and demonstrated the same genetic basis in the Canadian SARA family. For a peptide representing the SARA sequence, inhibition of anti-SARA against SARA-positive cells was 84.6% at a concentration of 1.0 mg/mL. CONCLUSION: We provide evidence that the SARA antigen is encoded by a SNV on GYPA and SARA has been reassigned to the MNS blood group system, now MNS47. This discovery provides a basis for application of genetic approaches in SARA typing when clinically indicated, for example, in HDFN.

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.

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.

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

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.

Leishmania in HLH: a rare finding with significant treatment implications.

Hemophagocytic lymphohistiocytosis (HLH) associated with visceral leishmaniasis (VL) is a very rare phenomenon. We report the first known North American case in a 21 month old boy. He was initially diagnosed with Epstein Barr virus (EBV) triggered HLH and treated with the international treatment protocol, HLH-2004. Stem cell transplant was planned due to repeated reactivations of disease, but his pretransplant bone marrow revealed an unexpected protozoan-Leishmania donovani. Treatment with liposomal amphotericin B led to resolution of all manifestations of HLH. We discuss how the clinical and laboratory features of both entities can closely mimic each other and are extremely difficult to differentiate. This case also raises the question of whether to screen all children with suspected HLH for Leishmania in a non endemic area.

Deep immunophenotypic analysis of the bone marrow progenitor cells in myelodysplastic syndromes.

BACKGROUND: Diagnosis of myelodysplastic syndromes (MDS) is often challenging and requires integration of clinical, morphologic, cytogenetics and molecular information. Flow cytometry immunophenotyping (FCIP) can support the diagnosis by demonstration of numerical and immunophenotypic abnormalities of progenitor and maturing myelomonocytic and erythroid populations. We have previously shown that comprehensive immunophenotypic analysis of the progenitor population is valuable in the diagnosis of MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). This study was designed to improve the analysis method and confirm its value in a larger cohort of patients. METHODS: FCIP of bone marrow samples from 105 patients with cytopenia(s) (with or without leukocytosis) and clinical concern for MDS or MDS/MPN was performed using a single-tube/10-color/13-marker assay. A modified analysis approach was used to obtain 11 progenitor parameters and 2 myelomonocytic parameters. RESULTS: Significantly higher number of abnormalities were identified in MDS and MDS/MPN cases when compared to cytopenic patients not meeting the diagnostic criteria for MDS (Non-MDS). A FCIP score that combined the 13 parameters showed a sensitivity of 89.8% and specificity of 93.5% for the diagnosis of MDS and MDS/MPN. The sensitivity was 100% for both MDS/MPN and higher-risk MDS, and 81.3% for lower-risk MDS. CONCLUSION: This study confirms that detailed immunophenotypic analysis of the progenitor population is powerful in the diagnosis of MDS and MDS/MPN. The combination of markers used in the panel allowed for evaluation of two relatively new parameters, namely myeloid progenitor heterogeneity and stem cell aberrancy, which improved the sensitivity of the assay for lower-risk MDS.

Exploring TBL1XR1 and NCOR1 Expression in B-cell Lymphoma Subtypes: Interaction With DNA Damage Repair Genes.

BACKGROUND/AIM: B-cell lymphomas are characterized by diverse genetic anomalies affecting B-cell differentiation. To expand targeted therapies, an in-depth grasp of the molecular dynamics in the germinal center (GC) is vital. Transducin ß-like 1 X-linked receptor 1 (TBL1XR1) and nuclear receptor corepressor 1 (NCOR1) are instrumental within the GC, modulating myriad oncogenic pathways. Their prognostic roles in various cancers are established, yet their precise impact on B-cell lymphoma is elusive. MATERIALS AND METHODS: Digital RNA quantification (Nanostring) of previously curated 188 B-cell lymphoma specimens across four subtypes, follicular lymphoma (FL), diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS), primary testicular lymphoma (PTL), and plasmablastic lymphoma (PBL), was reanalyzed with focus on TBL1XR1 and NCOR1 expression, juxtaposing them with 730 ontogenically linked genes. RESULTS: Notably, TBL1XR1 expression was significantly elevated in the PTL- ABC-subtype versus DLBCL-NOS- ABC-subtype (p<0.001), with no marked disparity in GCB-subtypes between them. The median TBL1XR1 expression was remarkably diminished in FL, yet, intriguingly, GCB-subtypes of DLBCL-NOS exhibited significantly enhanced expression compared to FL (p=0.001). In contrast, NCOR1's expression trajectory was consistent across DLBCL-NOS, PTL, and PBL. A strong inverse correlation between TBL1XR1 and NCOR1 was observed in PBL (p=0.001). Importantly, TBL1XR1's pronounced association with several DNA Damage repair (DDR) genes was noted suggesting influence on DNA repair. TBL1XR1-DDR gene signature was further validated employing a public data set of DLBCL-NOS. CONCLUSION: Our exploratory findings unravel the expression patterns of TBL1XR1/NCOR1 in B-cell lymphoma variants. The TBL1XR1-DDR genes connection offers insights into potential DNA repair roles, paving avenues for innovative therapies in B-cell lymphomas.

NMR-based metabolomic profiling can differentiate follicular lymphoma from benign lymph node tissues and may be predictive of outcome.

Follicular lymphoma (FL) is a cancer of B-cells, representing the second most common type of non-Hodgkin lymphoma and typically diagnosed at advanced stage in older adults. In contrast to the wide range of available molecular genetic data, limited data relating the metabolomic features of follicular lymphoma are known. Metabolomics is a promising analytical approach employing metabolites (molecules < 1 kDa in size) as potential biomarkers in cancer research. In this pilot study, we performed proton nuclear magnetic resonance spectroscopy (1H-NMR) on 29 cases of FL and 11 control patient specimens. The resulting spectra were assessed by both unsupervised and supervised statistical methods. We report significantly discriminant metabolomic models of common metabolites distinguishing FL from control tissues. Within our FL case series, we also report discriminant metabolomic signatures predictive of progression-free survival.

Exploring blast composition in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms: CD45RA and CD371 improve diagnostic value of flow cytometry through assessment of myeloblast heterogeneity and stem cell aberrancy.

BACKGROUND: Flow cytometry immunophenotyping (FCIP) can improve diagnosis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), although its application is challenging due to difficulties in standardization, complexity of antibody panels and subjective interpretation of data. Since blasts are invariably affected in these disorders, we developed a FCIP approach for detailed and objective analysis of the blast population. METHODS: FCIP using a one-tube 10-color (13-marker) antibody panel was performed on bone marrow samples from 23 MDS and 8 MDS/MPN patients, 21 cytopenic patients non-diagnostic for MDS (Non-MDS), and 16 Control samples. RESULTS: MDS and MDS/MPN cases demonstrated one to several immunophenotypic abnormalities including: increased myeloblasts, decreased stage-1 hematogones, aberrant stem cells, abnormal myeloblast heterogeneity/divergence from normal, increased or decreased CD45 intensity, increased CD117 or CD123 intensity, decreased CD38 intensity, and aberrant expression of lineage markers (CD5, CD19, CD56). A Blast score was developed that showed sensitivity of 80.6% and specificity of 90.5% for immunophenotypic diagnosis of MDS and MDS/MPN. Expression levels of CD45RA and CD371 were used to evaluate abnormal myeloblast heterogeneity and stem cell aberrancy. Both these features were, for the first time, incorporated into a scoring system and resulted in 19% increase in the sensitivity of the assay for lower-risk MDS. CONCLUSION: Deep immunophenotypic analysis of the blast population is valuable for diagnosis of MDS and MDS/MPN and can potentially provide sensitivity and specificity figures comparable to those previously described using more comprehensive panels that assess maturing myelomonocytic and erythroid elements in addition to progenitor cells.

Impaired natural killer cell counts and cytolytic activity in patients with severe COVID-19.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-driven coronavirus disease 2019 (COVID-19) has caused unprecedented human death and has seriously threatened the global economy. Early data suggest a surge in proinflammatory cytokines in patients with severe COVID-19, which has been associated with poor outcomes. We recently postulated that the inflammatory response in patients with severe COVID-19 disease is not inhibited by natural killer (NK) cells, resulting in a "cytokine storm." Here, we assessed the NK-cell functional activity and the associated cytokines and soluble mediators in hospitalized COVID-19 patients. Significantly impaired NK-cell counts and cytolytic activity were observed in COVID-19 patients when compared with healthy controls. Also, cytokines like interleukin 12 (IL12), IL15, and IL21 that are important for NK-cell activity were not detected systematically. Serum concentrations of soluble CD25 (sCD25)/soluble IL2 receptor α (sIL2-Rα) were significantly elevated and were inversely correlated with the percentage of NK cells. Impaired NK-cell cytolytic activity together with other laboratory trends including elevated sCD25 were consistent with a hyperinflammatory state in keeping with macrophage-activation syndrome. Our findings suggest that impaired counts and cytolytic activity of NK cells are important characteristics of severe COVID-19 and can potentially facilitate strategies for immunomodulatory therapies.

Distinction of hepatocellular adenoma from hepatocellular carcinoma with and without cirrhosis using E-cadherin and matrix metalloproteinase immunohistochemistry.

We studied the tumor cell expression patterns of E-cadherin and matrix metalloproteinase-1, -2, -7, and -9 in a tissue microarray composed of 20 normal livers, 10 hepatocellular adenomas, 43 hepatocellular carcinomas with cirrhosis and 33 hepatocellular carcinomas without cirrhosis. Hepatocellular adenoma was characterized by the complete absence of matrix metalloproteinase-7 expression; hepatocellular carcinoma with cirrhosis was characterized by a significantly low expression of E-cadherin; and hepatocellular carcinoma without cirrhosis was characterized by low matrix metalloproteinase-9 expression. The staining intensity score of E-cadherin=3, matrix a metalloproteinase-7<1, and matrix metalloproteinase-9>or=2 can be used as the diagnostic criteria for hepatocellular adenoma and for distinguishing hepatocellular adenoma from normal, hepatocellular carcinoma with cirrhosis, and hepatocellular carcinoma without cirrhosis. E-cadherin<2 and matrix metalloproteinase-9<2 can be used for distinguishing both hepatocellular carcinoma with cirrhosis and hepatocellular carcinoma without cirrhosis from normal. Although statistically not significant, hepatocellular carcinoma without cirrhosis showed a higher E-cadherin expression and a lower matrix metalloproteinase-9 expression than hepatocellular carcinoma with cirrhosis, which could be partially responsible for the less aggressive behavior found in hepatocellular carcinoma without cirrhosis when compared with hepatocellular carcinoma with cirrhosis. These results, if confirmed in a further study of small biopsy specimens and of histologically ambiguous cases, could lead to the application of these markers in the diagnosis and differential diagnosis of hepatocellular neoplasms in our surgical pathology practice.