Point Mutation Specific Antibodies in B-Cell and T-Cell Lymphomas and Leukemias: Targeting IDH2, KRAS, BRAF and Other Biomarkers RHOA, IRF8, MYD88, ID3, NRAS, SF3B1 and EZH2.
Diagnostics (Basel, Switzerland)
2021; 11 (4)
B-cell and T-cell lymphomas and leukemias often have distinct genetic mutations that are diagnostically defining or prognostically significant. A subset of these mutations consists of specific point mutations, which can be evaluated using genetic sequencing approaches or point mutation specific antibodies. Here, we describe genes harboring point mutations relevant to B-cell and T-cell malignancies and discuss the current availability of these targeted point mutation specific antibodies. We also evaluate the possibility of generating novel antibodies against known point mutations by computationally assessing for chemical and structural features as well as epitope antigenicity of these targets. Our results not only summarize several genetic mutations and identify existing point mutation specific antibodies relevant to hematologic malignancies, but also reveal potential underdeveloped targets which merit further study.
View details for DOI 10.3390/diagnostics11040600
View details for PubMedID 33801781
A Review of Genetic Abnormalities in Unicentric and Multicentric Castleman Disease.
2021; 10 (4)
Castleman disease (CD) is a rare lymphoproliferative disorder known to represent at least four distinct clinicopathologic subtypes. Large advancements in our clinical and histopathologic description of these diverse diseases have been made, resulting in subtyping based on number of enlarged lymph nodes (unicentric versus multicentric), according to viral infection by human herpes virus 8 (HHV-8) and human immunodeficiency virus (HIV), and with relation to clonal plasma cells (POEMS). In recent years, significant molecular and genetic abnormalities associated with CD have been described. However, we continue to lack a foundational understanding of the biological mechanisms driving this disease process. Here, we review all cases of CD with molecular abnormalities described in the literature to date, and correlate cytogenetic, molecular, and genetic abnormalities with disease subtypes and phenotypes. Our review notes complex karyotypes in subsets of cases, specific mutations in PDGFRB N666S in 10% of unicentric CD (UCD) and NCOA4 L261F in 23% of idiopathic multicentric CD (iMCD) cases. Genes affecting chromatin organization and abnormalities in methylation are seen more commonly in iMCD while abnormalities within the mitogen-activated protein kinase (MAPK) and interleukin signaling pathways are more frequent in UCD. Interestingly, there is a paucity of genetic studies evaluating HHV-8 positive multicentric CD (HHV-8+ MCD) and POEMS-associated CD. Our comprehensive review of genetic and molecular abnormalities in CD identifies subtype-specific and novel pathways which may allow for more targeted treatment options and unique biologic therapies.
View details for DOI 10.3390/biology10040251
View details for PubMedID 33804823
Measurement of Cell Volume Using In-Line Digital Holography.
Microscopy (Oxford, England)
The measurement of the volume of blood cells is important for clinical diagnosis and patient management. While digital holography microscopy (DHM) has been used to obtain such information, previous off-axis setups usually involve a separated reference beam and are thus not very easy to implement. Here, we use the simple in-line Gabor setup without separation of a reference beam to measure the shape and volume of cells mounted on glass slides. Inherent to the in-line holograms, the reconstructed phase of the object is affected by the virtual image noise, producing errors in the cell volume measurement. We optimized our approach to use a single hologram without phase retrieval, increasing distance between cell and hologram plane to reduce the measurement error of cell volume to less than 6% in some instances. Therefore, the in-line Gabor setup can be a useful and simple tool to obtain volumetric and morphologic cellular information.
View details for DOI 10.1093/jmicro/dfaa077
View details for PubMedID 33372674
Hypodiploid B-Lymphoblastic Leukemia Presenting as an Isolated Orbital Mass Prior to Systemic Involvement: A Case Report and Review of the Literature.
Diagnostics (Basel, Switzerland)
2020; 11 (1)
We describe a 4-year-old boy who presented with progressive right periorbital edema and proptosis, with no systemic symptoms, who was found to have B-lymphoblastic leukemia (B-ALL). Magnetic resonance imaging (MRI) showed an enhancing mass centered in the right superolateral extraconal orbit. Orbital biopsy was consistent with B-ALL (CD99, TdT, LCA cocktail, CD34, CD79, CD10, PAX5, MIB1 positive; CD3, CD20 negative). A subsequent bone marrow aspirate confirmed a diagnosis of B-ALL with 80% blasts by flow cytometry and haploid cytogenetic findings. The patient improved clinically after chemotherapy. There are seven cases previously reported in the literature with hematogenous orbital masses at initial presentation of childhood ALL, but all with systemic symptoms or an abnormal complete blood count (CBC) at presentation. Our case is the first report in which an orbital mass preceded detectable systemic or laboratory evidence of ALL. This patient highlights the importance of differentiating benign causes of eyelid swelling from malignant ones.
View details for DOI 10.3390/diagnostics11010025
View details for PubMedID 33375646
Histology-Independent Signature Distinguishes Kikuchi-Fujimoto Disease/Systemic Lupus Erythematosus-Associated Lymphadenitis From Benign and Malignant Lymphadenopathies.
American journal of clinical pathology
Kikuchi-Fujimoto disease (KFD) and systemic lupus erythematosus (SLE) are benign entities with histologic features that raise concern about malignancy and infection. We searched for a histology-independent KFD/SLE signature relying on only immunophenotype and basic clinical characteristics.A histology-independent KFD/SLE signature was generated using 975 excised lymph nodes with flow immunophenotyping, including 16 cases of KFD/SLE. This signature was then evaluated in 1,198 fine-needle aspiration (FNA) specimens.The top flow cytometry discriminant for KFD/SLE was uniform CD38+ expression on CD19+ events. Immunohistochemistry demonstrated nodules of IgD+, IgM- B cells surrounding necrotizing and activated T-cell areas. A signature combining 6 flow cytometry criteria with age and sample site had a positive predictive value of 88% for KFD/SLE, which had a prevalence of 1.6%. All 4 signature-positive FNA cases with follow-up excision were KFD/SLE. At a second institution, 4 of 5 KFD/SLE cases passed the top discriminant.A flow cytometry signature combined with age and biopsy site identifies KFD/SLE independent of histology, suggesting a shared immune composition and independently confirming that KFD/SLE represents a distinct entity. Unexpectedly, an IgD+CD38+ small B-cell population is a distinctive feature of KFD/SLE, suggesting a possible pathologic role for anergic/autoreactive B cells.
View details for DOI 10.1093/ajcp/aqaa036
View details for PubMedID 32367142
A meta-analysis of SARS-CoV-2 patients identifies the combinatorial significance of D-dimer, C-reactive protein, lymphocyte, and neutrophil values as a predictor of disease severity.
International journal of laboratory hematology
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), known to be the causative agent of COVID-19, has led to a worldwide pandemic. At presentation, individual clinical laboratory blood values, such as lymphocyte counts or C-reactive protein (CRP) levels, may be abnormal and associated with disease severity. However, combinatorial interpretation of these laboratory blood values, in the context of COVID-19, remains a challenge.To assess the significance of multiple laboratory blood values in patients with SARS-CoV-2 and develop a COVID-19 predictive equation, we conducted a literature search using PubMed to seek articles that included defined laboratory data points along with clinical disease progression. We identified 9846 papers, selecting primary studies with at least 20 patients for univariate analysis to identify clinical variables predicting nonsevere and severe COVID-19 cases. Multiple regression analysis was performed on a training set of patient studies to generate severity predictor equations, and subsequently tested on a validation cohort of 151 patients who had a median duration of observation of 14 days.Two COVID-19 predictive equations were generated: one using four variables (CRP, D-dimer levels, lymphocyte count, and neutrophil count), and another using three variables (CRP, lymphocyte count, and neutrophil count). In adult and pediatric populations, the predictive equations exhibited high specificity, sensitivity, positive predictive values, and negative predictive values.Using the generated equations, the outcomes of COVID-19 patients can be predicted using commonly obtained clinical laboratory data. These predictive equations may inform future studies evaluating the long-term follow-up of COVID-19 patients.
View details for DOI 10.1111/ijlh.13354
View details for PubMedID 33010111
A comprehensive analysis of RHOA mutation positive and negative angioimmunoblastic T-cell lymphomas by targeted deep sequencing, expression profiling and single cell digital image analysis.
International journal of molecular medicine
Angioimmunoblastic T‑cell lymphoma (AITL) is a uniquely aggressive mature T‑cell neoplasm. In recent years, recurrent genetic mutations in ras homolog family member A (RHOA), tet methylcytosine dioxygenase 2 (TET2), DNA methyltransferase 3 alpha (DNMT3A) and isocitrate dehydrogenase [NADP(+)] 2 (IDH2) have been identified as associated with AITL. However, a deep molecular study assessing both DNA mutations and RNA expression profile combined with digital image analysis is lacking. The present study aimed to evaluate the significance of molecular and morphologic features by high resolution digital image analysis in several cases of AITL. To do so, a total of 18 separate tissues from 10 patients with AITL were collected and analyzed. The results identified recurrent mutations in RHOA, TET2, DNMT3A, and IDH2, and demonstrated increased DNA mutations in coding, promoter and CCCTC binding factor (CTCF) binding sites in RHOA mutated AITLs vs. RHOA non‑mutated cases, as well as increased overall survival in RHOA mutated patients. In addition, single cell computational digital image analysis morphologically characterized RHOA mutated AITL cells as distinct from cells from RHOA mutation negative patients. Computational analysis of single cell morphological parameters revealed that RHOA mutated cells have decreased eccentricity (more circular) compared with RHOA non‑mutated AITL cells. In conclusion, the results from the present study expand our understanding of AITL and demonstrate that there are specific cell biological and morphological manifestations of RHOA mutations in cases of AITL.
View details for DOI 10.3892/ijmm.2020.4686
View details for PubMedID 32945366
LMO2 Expression Distinguishes T-Lymphoblastic Leukemia/Lymphoma from Indolent T-Lymphoblastic Proliferations.
An indolent T-lymphoblastic proliferation (iT-LBP) is a benign, reactive expansion of immature TdT+ T-cells found in extrathymic tissues. iT-LBP can be challenging to distinguish from malignant processes, specifically T-lymphoblastic lymphoma (T-LBL), given the overlapping clinical and histologic features. Recently, it has been shown that LIM domain only 2 (LMO2) is overexpressed in T-LBL but not in reactive immature TdT+ T-cells in the thymus. Based on these findings, we investigated the expression of LMO2 by immunohistochemistry and its role in differentiating iT-LBPs from T-LBLs.We retrospectively identified cases of iT-LBP and T-LBL from the pathology archives of four institutions. Seven cases of iT-LBP (including five new cases that have not been reported in the literature) and 13 cases of T-LBL were analyzed. Clinical, morphologic, immunophenotypic and molecular data were analyzed. Immunohistochemical staining with LMO2 was performed on all cases of iT-LBP and T-LBL.A review of five new cases of iT-LBP showed similar morphologic, immunophenotypic and molecular features to prior reported cases. All cases of iT-LBP were negative for LMO2 (0/7) while 92% of T-LBL cases (12/13) expressed LMO2; sensitivity 92% (confidence interval 64-100%) and specificity 100% (confidence interval 59-100%).We confirm prior published findings that cases of iT-LBPs demonstrate highly overlapping morphologic and immunophenotypic features when compared to T-LBL. Importantly, expression of LMO2 is a sensitive and specific marker to rule out iT-LBP.
View details for DOI 10.1111/his.14176
View details for PubMedID 32526041
A Long-Term Study of Persistent Sézary Syndrome: Evidence for Antigen Shift by Multiparameter Flow Cytometry and Its Significance in Overall Survival.
The American Journal of dermatopathology
2020; 42 (6): 389–96
Sézary syndrome (SS) is a peripheral T-cell lymphoma characterized by erythroderma, diffuse lymphadenopathy, and circulating neoplastic T cells, which classically show a helper T-cell immunophenotype with loss of CD7 and CD26. Flow cytometry is often used to identify and enumerate populations of Sézary cells in the peripheral blood; however, the significance and frequency of antigen shift over time is unclear. In this article, we follow the immunophenotype of the neoplastic T-cell population from 28 patients with SS across 415 flow cytometry studies. Antigen shift for each patient was assigned as none, minimal = 1-2 markers by 1°, moderate = up to 3 markers, or marked ≥ 4 markers. Sixty-four percent (18/28) of patients showed antigen shift, and among those with antigen shift, the majority showed minimal (8/18) or moderate antigen shift (7/18) with fewer demonstrating marked shift (3/18). Patients without antigen shift showed a trend toward improved overall survival in comparison with patients demonstrating any degree of antigen shift. Antigen shift is seen in a significant proportion of cases of SS with long-term follow-up and may be a marker of more aggressive disease.
View details for DOI 10.1097/DAD.0000000000001637
View details for PubMedID 32433315
- Flow Cytometry Signature for Kikuchi-Fujimoto/Lupus Lymphadenitis Derived From 975 Benign and Malignant Lymphadenopathies OXFORD UNIV PRESS INC. 2019: S105–S106
Indolent In Situ B-Cell Neoplasms With MYC Rearrangements Show Somatic Mutations in MYC and TNFRSF14 by Next-generation Sequencing.
The American journal of surgical pathology
Systemic high-grade B-cell lymphomas (HGBCLs) with MYC gene rearrangements are clinically aggressive. In situ lesions with indolent behavior have not been described to date. We have identified 2 cases of in situ B-cell neoplasms with MYC rearrangements (IS-BCN, MYC) occurring, and focally confined to ≤4 lymphoid follicles in otherwise healthy individuals and without clinical progression despite minimal intervention (surgical only). Morphologically similar to systemic HGBCLs, the low power view of these lesions showed a starry sky pattern with numerous mitotic figures. High power imaging demonstrated these cells to be medium-large in size with irregular nuclear contours, immature chromatin, and prominent nucleoli. Immunophenotypically these cells were light chain restricted, positive for CD20, CD10, c-Myc, and dim or negative for BCL2 with a Ki67 proliferative index of >95%. By fluorescence in situ hybridization studies, we detected MYC translocations in these cells but no rearrangements in BCL2 or BCL6. Microdissection of neoplastic cells in these patients followed by targeted next-generation sequencing identified a mutation in MYC, D2N, and an indel in TNFRSF14. Mutations in ID3 or TCF3 were not identified. Although rare, these lesions should be separated from HGBCLs involving follicles but with systemic spread which has been previously described. Unlike systemic lymphomas with MYC gene rearrangements, these in situ B-cell neoplasms with MYC rearrangements did not require systemic therapy and no progression has been seen in either patient beyond 1 year (29 and 16mo). Our work offers pathologic and biologic insight into the early process of B-cell neoplasia.
View details for DOI 10.1097/PAS.0000000000001338
View details for PubMedID 31368914
The Significance of Dim Cytoplasmic CD3 Expression in Acute Myeloid Leukemia: A Long-Term Retrospective Study Identifies an Association with Acute Promyelocytic Leukemia with FLT3-ITD Mutations
NATURE PUBLISHING GROUP. 2018: 529
View details for Web of Science ID 000429308603354
Noninvasive Assessment of Gene Transfer and Expression by In Vivo Functional and Morphologic Imaging in a Rabbit Tumor Model
2013; 8 (6)
To evaluate the importance of morphology in quantifying expression after in vivo gene transfer and to compare gene expression after intra-arterial (IA) and intra-tumoral (IT) delivery of adenovirus expressing a SSTR2-based reporter gene in a large animal tumor model.Tumor directed IA or IT delivery of adenovirus containing a human somatostatin receptor type 2A (Ad-CMV-HA-SSTR2A) gene chimera or control adenovirus (Ad-CMV-GFP) was performed in VX2 tumors growing in both rabbit thighs. Three days later, ¹¹¹In-octreotide was administered intravenously after CT imaging using a clinical scanner. ¹¹¹In-octreotide uptake in tumors was evaluated the following day using a clinical gamma-camera. Gene expression was normalized to tumor weight with and without necrosis. This procedure was repeated on nine additional rabbits to investigate longitudinal gene expression both 5 days and 2 weeks after adenovirus delivery. CT images were used to evaluate tumor morphology and excised tissue samples were analyzed to determine ¹¹¹In-octreotide biodistribution ex vivo.VX2 tumors infected with Ad-CMV-HA-SSTR2 had greater ¹¹¹In-octreotide uptake than with control virus (P<0.05). Intra-arterial and intra-tumoral routes resulted in similar levels of gene expression. Longitudinally, expression appeared to wane at 2 weeks versus 5 days after delivery. Areas of necrosis did not demonstrate significant uptake ex vivo. Morphology identified areas of necrosis on contrast enhanced CT and upon excluding necrosis, in vivo biodistribution analysis resulted in greater percent injected dose per gram (P<0.01) and corresponded better with ex vivo biodistribution(r = 0.72, P<0.01, Coefficient of the x-variable = .72) at 2 weeks than without excluding necrosis (P<0.01).Tumor specificity and high transgene expression can be achieved in tumors via both tumor directed intra-arterial and intra-tumoral delivery in a large animal tumor model. Using clinical machines, morphologic imaging contributes to functional imaging for quantifying SSTR2-based reporter expression in vivo.
View details for DOI 10.1371/journal.pone.0062371
View details for Web of Science ID 000320440500002
View details for PubMedID 23762226
View details for PubMedCentralID PMC3677885
Visualizing the Prostate Gland by MR Imaging in Young and Old Mice
2013; 8 (3)
Prostate imaging requires optimization in young and old mouse models. We tested which MR sequences and field strengths best depict the prostate gland in young and old mice; and, whether prostate MR signal, size, and architecture change with age.Magnetic resonance imaging (MRI) of the prostate of young (2 months) and old (18 months) male nude mice (n = 6) was performed at 4.7 and 7 T and SCID mice (n = 6) at 7 T field strengths, using T1, fat suppressed T1, DWI, T2, fat suppressed T2, as well as T2-based- and proton density-based Dixon "water only" sequences. Images were ranked for best overall sequence for prostate visualization, prostate delineation, and quality of fat suppression. Prostate volume and signal characteristics were compared and histology was performed.T2-based-Dixon "water only" images ranked best overall for prostate visualization and delineation as well as fat suppression (n = 6, P<0.001) at both 4.7 T and 7 T in nude and 7T in SCID mice. Evaluated in nude mice, T2-based Dixon "water only" had greater prostate CNR and lower fat SNR at 7 T than 4.7 T (P<0.001). Prostate volume was less in older than younger mice (n = 6, P<0.02 nude mice; n = 6, P<0.002 SCID mice). Prostate T2 FSE as well as proton density-based and T2-based-Dixon "water only" signal intensity was higher in younger than older mice (P<0.001 nude mice; P<0.01 SCID mice) both at 4.7 and 7 T. This corresponded to an increase in glandular hyperplasia in older mice by histology (P<0.01, n = 6).T2-based Dixon "water only" images best depict the mouse prostate in young and old nude mice at 4.7 and 7 T. The mouse prostate decreases in size with age. The decrease in T2 and T2-based Dixon "water only" signal with age corresponds with glandular hyperplasia. Findings suggest age should be an important determinant when choosing models of prostate biology and disease.
View details for DOI 10.1371/journal.pone.0055746
View details for Web of Science ID 000315897100007
View details for PubMedID 23469167
View details for PubMedCentralID PMC3585879
Involvement of microRNA181a in differentiation and cell cycle arrest induced by a plant-derived antioxidant carnosic acid and vitamin D analog doxercalciferol in human leukemia cells.
MicroRNA (Shariqah, United Arab Emirates)
2012; 1 (1): 26-33
1,25-dihydroxyvitamin D3 (1,25D) has been shown to influence differentiation, cell proliferation and cell death in cultured leukemia cells. However, its clinical use is limited by its hypercalcemic effects. An analog of 1,25D, doxercalciferol (1-D2), has anti-tumor activity, with markedly reduced calcemic effects, which makes it a potential agent for clinical treatment of AML. Previous studies suggested that the combination of 1,25D with other agents, such as plant-derived antioxidants, can have additive or synergistic anti-cancer activities in leukemia cells. Here we report that 1-D2 induced monocytic differentiation of HL60 and U937 cells, and that the antioxidant carnosic acid (CA) enhanced 1-D2 induced differentiation and cell cycle arrest. MicroRNA181a (miR181a) expression was also reduced after exposure to CA/1-D2. Since the cell cycle regulator p27Kip1 has been shown to be a target of miR181a, we modulated miR181a levels to determine if it plays a role in CA/1-D2 induced differentiation and cell cycle arrest in AML cells. We found that transfection of antisense miR181a potentiated CA/1-D2-induced cell differentiation, while the transfection of precursor of miR181a partially inhibited the effect of CA/1-D2 on the differentiation. These findings imply that miR181a has a role in CA/1-D2- induced differentiation and cell cycle arrest of HL60 and U937 cells, and shows a broader participation of miR181a in cell cycle control in leukemia cells.
View details for PubMedID 25048087