scholarly journals Flow Cytometry Can Diagnose Classical Hodgkin Lymphoma in Lymph Nodes With High Sensitivity and Specificity

2009 ◽  
Vol 131 (3) ◽  
pp. 322-332 ◽  
Author(s):  
Jonathan R. Fromm ◽  
Anju Thomas ◽  
Brent L. Wood
Keyword(s):  
Flow Cytometry ◽  
Lymph Nodes ◽  
Hodgkin Lymphoma ◽  
Sensitivity And Specificity ◽  
High Sensitivity ◽  
Classical Hodgkin Lymphoma

Identification and Purification of Hodgkin Cells from Lymph Nodes Involved by Classical Hodgkin Lymphoma by Flow Cytometry and Flow Cytometric Cell Sorting.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2262-2262
Author(s):  
Jonathan R. Fromm ◽  
Steven J. Kussick ◽  
Brent L. Wood
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Hodgkin Lymphoma ◽  
Cell Sorting ◽  
Classical Hodgkin Lymphoma ◽  
Tissue Sections ◽  
Hla Dr ◽  
Blocking Antibodies

Abstract The diagnosis of classical Hodgkin lymphoma (CHL) has historically been made in tissue sections, as attempts to identify the neoplastic Hodgkin and Reed-Sternberg (HRS) cells of CHL by flow cytometry (FC) have been largely unsuccessful. As HRS cells are known to be ringed (“rosetted”) by benign/reactive T cells, we hypothesized that in cell suspensions the HRS will be bound to T cells (forming T cell rosettes), and that consequently the rosettes would have a composite T-cell/HRS immunophenotype by FC (CD3+/CD15+/CD20−/CD30+/CD45+). We further hypothesized that specific antibodies to the adhesion molecules known to be involved in T cell/HRS cell binding (CD2 and LFA-1 on the T cell, and CD54 and CD58 on the HRS cell) might result in “naked” (unbound) HRS cells, enabling us to use FC to identify HRS cells with the expected immunophenotype (CD3−/CD15+/CD20−/CD30+/CD45−). Our initial FC studies of the HRS cell line L1236 demonstrated that CD15, CD30, CD40, CD71, CD86, CD95, and HLA-DR, but not CD3 or CD20, were brightly expressed on these cells and may be useful in identification of HRS in authentic cases of CHL involving lymph nodes. In mixing experiments, L1236 cells spontaneously bound normal T cells, analogous to T cell rosetting of HRS cells in CHL; these interactions could be blocked specifically using a cocktail of unlabeled antibodies to CD2, LFA-1, CD54, and CD58. Among 27 lymph nodes involved by CHL, this novel FC method, in which 250,000 to 500,000 total lymph node cells were evaluated, and in which up to ten cellular antigens were assessed simultaneously, enabled HRS cells to be identified in 89% of cases. 82% of these cases demonstrated interactions between HRS cells and T cells that could be disrupted with blocking antibodies. None of 29 non-CHL neoplasms, and none of 23 reactive lymph nodes, demonstrated HRS populations by FC. The proportions of cases where the HRS population showed expression of CD15, CD30, CD40, CD71, CD86, CD95, and HLA-DR, and absence of CD3 and CD20 was similar to that described previously in tissue sections by immunohistochemistry. Interestingly, in contrast to the findings in tissue sections, by FC the non-rosetted HRS cells of most CHL cases (73%) demonstrated detectable expression of CD45, usually at a low level. Finally, cell sorting experiments confirmed that (1) populations identified by FC have the cytomorphology of HRS cells, (2) HRS/T cell rosettes can be detected by FC, and (3) these rosettes can disrupted by the blocking antibody cocktail. This FC technique offers a potential alternative to immunohistochemistry in confirming the diagnosis of CHL and, through cell sorting, provides a means of rapidly purifying HRS cells.

Flow cytometry CD4+CD26−CD38+ lymphocyte subset in the microenvironment of Hodgkin lymphoma-affected lymph nodes

2014 ◽  
Vol 93 (8) ◽  
pp. 1319-1326 ◽  
Author(s):  
Rosa Di Gaetano ◽  
Valentina Gasparetto ◽  
Andrea Padoan ◽  
Barbara Callegari ◽  
Laura Candiotto ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Lymph Nodes ◽  
Hodgkin Lymphoma ◽  
Lymphocyte Subset

scholarly journals Nodular Lymphocyte Predominant Hodgkin Lymphoma - A Retrospective Immunohistochemical Study of Patients in Bangalore, Karnataka

2021 ◽  
Vol 8 (23) ◽  
pp. 1966-1969
Author(s):  
Shankar Anand ◽  
Akshatha C ◽  
Libin Babu Cherian ◽  
Ramachandra C
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Hodgkin Lymphoma ◽  
Classical Hodgkin Lymphoma ◽  
Double Positive ◽  
Cervical Lymph Nodes ◽  
Histiocytic Cell ◽  
Immunohistochemical Markers ◽  
Lp Cells

BACKGROUND The term Hodgkin’s lymphoma includes classical Hodgkin lymphoma (CHL) and the less common nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). NLPHL is a B cell neoplasm usually characterised by nodular or follicular and diffuse proliferation of small lymphocytes with single scattered large neoplastic cells (LP/L&H/Popcorn cells). NLPHL accounts for 10 % of all Hodgkin lymphoma. METHODS This is a retrospective study. Histopathology slides and blocks of 24 cases of nodular lymphocyte predominant Hodgkin lymphoma were collected from the archives of histopathology from 2011 to 2015. The immunohistochemistry slides of the corresponding histopathology cases were also assembled. Both the slides were reviewed by three expert onco-pathologists and IHC markers were studied and compared. RESULTS Patients were mostly young between 20 and 40 years (16 / 24, 66.67 %). There was a distinct male preponderance (20 / 24, 83.3 %). Most cases involved cervical, axillary or inguinal lymph nodes, with cervical lymph nodes being the most common (13 / 24, 54 %). It was found that CD45, CD20, CD79a and PAX5 staining highlighted the LP cells in all twenty-four cases, while OCT - 2 and BOB - 1 were highlighted in twenty-three cases (95.8 %), which was statistically significant. CD3 and CD5 IHC staining on T cell rosettes and background reactive T cells were examined, and it was seen that CD3 expression was far more consistent than CD5 expression in T cell rosettes and reactive T cells. Also, it was seen that, those cases which were double positive for CD3 and CD5 constitutes only eight cases (8 / 24, 33.3 %). CONCLUSIONS CD3 is a more consistent marker than CD5 in demonstrating surrounding reactive T cells in NLPHL. CD45, PAX5, CD20, BOB - 1 and OCT - 2 are consistent immunohistochemical markers of LP cells. KEYWORDS Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL), Classical Hodgkin Lymphoma (CHL), Cluster Differentiation (CD), Lymphocyte Predominant Cells (LP Cells), Lymphocyte and Histiocytic Cell (L & H Cell)

Regulation of Jak2 Kinase Gene by the Mir-135 Family in Classical Hodgkin Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 518-518
Author(s):  
Alfons Navarro ◽  
Tania Diaz ◽  
Antonio Martinez ◽  
Anna Gaya ◽  
Aina Pons ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Cell Line ◽  
Hodgkin Lymphoma ◽  
Disease Free Survival ◽  
Mirna Precursor ◽  
Negative Control ◽  
Classical Hodgkin Lymphoma ◽  
Protein Levels ◽  
Post Transfection ◽  
Specific Subset

Abstract Alterations of JAK/STAT signaling molecules have been reported in several lymphomas, including classical Hodgkin lymphoma (cHL). Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis, with Jak kinase overexpression promoting in vitro cell transformation. JAk2 is a cytoplasmic tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. Activating mutation has been found in myeloid neoplasms but the expression of JAK2 in primary mediastinal large B-cell lymphomas and classical Hodgkin lymphomas is not due to mutations. On the other hand, miRNAs play an important role in cHL, where they regulate the expression of genes that are crucial for the transformation process and to rescue Hodgkin/Reed Sternberg cells from apoptosis. We have previously shown that miR-135a, a putative miRNA involved in JAK2 expression, is present in the miRNA signature of cHL. The aims of the study were to analyze the role of miR-135a in Hodgkin cells and to determine if Jak2 is a target gene of this miRNA. Moreover, we examined whether the expression of miR-135a in diagnostic samples predicted clinical outcome in cHL. To that aim, 50nM and 100nM of pre-miR-135 and pre-miRNA precursor Negative Control (Ambion) were transfected in the L-428 cHL cell line using nucleofection (AMAXA, Solution L and program X-001). MiR-135a levels were analyzed by stem-loop-RT-PCR and Real-time (TaqMan MicroRNA Assays, Applied Biosystems) in a 7500 Sequence Detection system. Apoptosis levels were assayed by Caspase-Glo 3/7 luminescent assay that measures caspase-3 and -7 activities. Jak2 protein levels were analyzed by Western Blot (Abcam moAb). Eighty-nine adult patients (median age, 29 yrs [range, 13–89]; males 47%) diagnosed with cHL at a single institution between September 1995 and June 2005 were studied. Seven patients (7.8%) were HIV+. Histological subtypes: nodular sclerosis (79%) and mixed cellularity (21%). Epstein-Barr Virus (EBV) was present in 28% of the samples. First-line treatment consisted of ABVD type therapies. Total RNA was extracted from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation (Ambion). Statistical analysis was performed with SPSS 14.0. Clinical outcomes analyzed were relapse rate and disease-free survival (DFS). Of 89 patients, 75 (84.3%) achieved CR, 7 (7.8%) PR, while 7 (7.8%) were chemoresistant. After a median follow-up of 43 months (range, 1–128), overall survival was 83% and DFS 74%. Results showed that miR-135a was underexpressed in the cHL L-428 cell line and in cHL samples compared to reactive lymph nodes. After pre-miR-135 transfection, the miR-135a levels in L-428 cells increased 12 Ct at 24h. Analysis at 24 and 48 hours post-transfection showed a proportion of apoptotic cells 20% and 30% higher than in control cells (pre-miRNA precursor Negative control). The analysis of Jak2 protein levels at 48 and 72 hours post-transfection showed a dose-dependent reduction of the protein expression of 28%(100nM-48h) and 58%(100nM-72h). The proliferation assay showed that pre-miR-135-transfected cells grew less than control cells transfected with pre-miRNA precursor negative control. All these results are along the same line as those found in primary tumor. Patients could be divided into two groups according to miR-135a expression: high and low expression. Patients with low miR-135a expression had a higher probability of relapse than those with high miR-135a expression (p=0.045). In the multivariate analysis, only miR-135a expression emerged as a prognostic factor for relapse (RR=6.533, p=0.021). In conclusion, miR-135a downregulation seems to play a role in the rescue of Hodgkin/Reed Sternberg cells from apoptosis and influence the transformation event through the regulation of Jak2 levels. In addition, expression levels of miR-135a may be a prognostic marker for risk of relapse in cHL patients.

Prognostic Significance Of a 4-Microrna Signature Targeting JAK2 In Classical Hodgkin Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 629-629 ◽  
Author(s):  
Alfons Navarro ◽  
Anna Gaya ◽  
Anna Cordeiro ◽  
Blanca Gonzalez-Farre ◽  
Marina Díaz-Beyá ◽  
...  
Keyword(s):  
Western Blot ◽  
Lymph Nodes ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Renilla Luciferase ◽  
Luciferase Assay ◽  
Prognostic Impact ◽  
Classical Hodgkin Lymphoma ◽  
Expression Levels ◽  
Low Expression

Abstract Introduction The behavior of classical Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the Hodgkin/Reed Sternberg (HRS) cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. One of the genetic lesions most frequently found in HRS cells involves members of the JAK/STAT signaling pathway. However, few studies have identified microRNAs (miRNAs) that target JAK2 in cHL. In a previous study, our group identified miR-135a as a JAK2 regulator and showed the prognostic impact of this miRNA in a small set (n=89) of cHL patients (Navarro A. et al. Blood 2009). In the present study, we have examined novel miRNAs targeting JAK2 and evaluated the prognostic impact of their expression levels in 170 lymph nodes of cHL patients. Methods TargetScan and miRbase were used to identify JAK2-related miRNAs. The conserved miRNAs with higher scores were selected and further validated by Renilla-Luciferase assay and Western Blot. We performed a Renilla-Luciferase assay with a modified expression vector psiChecK-2 containing the complete 3’UTR region of JAK2 cloned in the 3’UTR region of Renilla luciferase gene. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in the HDMYZ cHL cell line together with 1μM of modified psicheck2 vector using Lipofectamine 2000, and luminescence was read at 24 hours. Further validation of the miRNA target was performed by Western Blot in three cHL cell lines (L428, L-1236 and HDMYZ). The expression levels of the miRNAs identified as targeting JAK2 were then assessed in lymph nodes from 170 cHL patients (median age, 33 years; male, 51%; EBV, 43.8%; HIV, 12%) diagnosed and treated in a single institution. Moreover, 15 reactive lymph nodes (RLN) were used as normal controls. RNA was purified from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation kit. The miRNA expression was analyzed using TaqMan microRNA assays. Statistical analyses were performed using R v2.13 and PASW Statistics 18. Results The bioinformatic analysis identified seven microRNAs as possible regulators of JAK2 (miR-34a, miR-101, miR-135a, miR-204, miR-216a, miR-216b and miR-375). The Renilla-Luciferase assay confirmed that in addition to miR-135a, miR-101 (61%; p=0.01), miR-204 (46%; p=0.003) and miR-216a (64.8%; p=0.02) also targeted JAK2. These findings were confirmed by Western Blot analysis of JAK2 levels after transfection with pre-miRNAs (median % of protein reduction in the 3 cell lines of 21.1%, 46.7% and 24%, respectively). The analysis of these miRNAs in RLN showed that miR-101, miR-135a and miR-204 were significantly downregulated in lymph nodes of cHL patients (p<0.001, p<0.001 and p=0.02 respectively) in comparison with RLN used as control. In contrast, miR-216 did not show significant differences in comparison with RLN. Interestingly, the analysis of the expression levels of these 4 miRNA in the patient lymph nodes had an impact on prognosis. First, low expression levels of miR-135a was associated with lower rate of complete response to first line treatment (76% vs 89%, p=0.012) and a trend was observed for miR-216(p=0.077). Secondly, patients with low expression levels of miR-101 or miR-204 had shorter disease-free survival (DFS) (151 vs 109 months, p=0.020; and 149.2 vs 105.5 months, p=0.055). Finally, patients with low levels of miR-135a, miR-204 or miR-216 had shorter overall survival (OS) (158 vs 136 months; p=0.039; and 157.5 vs 110 months, p=0.025; and 165 vs 140 moths, p=0.011 respectively). Since all four miRNAs showed prognostic impact (DFS, OS or treatment response), we decide to analyze the prognostic impact of the combination of the 4 miRNAs. Patients were then classified in two subgroups according to the expression levels of all four miRNAs. Patients with low expression of more than 2 miRNAs had shorter OS (163 vs 132 months; p=0.012). The multivariate analysis identified the combination of the four miRNAs as an independent prognostic marker of OS (OR, 7.6; 95% CI, 1.1-57.2; p=0.048) together with Age≥45years (OR, 5.9; 95% CI, 2.4-14.4; p<0.001) and B symptoms (OR, 2.6; 95% CI, 1.09-6.3; p=0.03). Conclusions MiR-101, miR-135a, miR-204 and miR-216 regulate JAK2 and are independent prognostic factors in cHL. Acknowledgments SDCSD of School of Medicine of UB. AC is an APIF-UB fellow. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Rare Case of Classical Hodgkin Lymphoma of the Thyroid Gland

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A879-A879
Author(s):  
Chandriya Chandran ◽  
Janna Prater ◽  
Jane Valerie Mayrin ◽  
Nancy A Young ◽  
Andrew Lytle ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ct Scan ◽  
Hodgkin Lymphoma ◽  
Patient Flow ◽  
Needle Aspiration ◽  
Neck Mass ◽  
Lymphoid Cells ◽  
Mediastinal Lymphadenopathy ◽  
Classical Hodgkin Lymphoma ◽  
Rapid Improvement

Abstract Background: Hodgkin lymphoma (HL) classically occurs in the lymph nodes and only in about 5% of cases occurs in extra-nodal sites. Primary thyroid lymphomas, most of which are non-Hodgkin lymphomas, comprise less than 5% of all thyroid malignancies. Only a few cases of HL of the thyroid have been reported in the literature, and in most of these cases, fine-needle aspiration biopsy (FNAB) was unreliable for diagnosis. We present a case of classical Hodgkin lymphoma of the thyroid that was falsely negative from both FNAB and core needle biopsy specimens and was diagnosed after surgery. Clinical Case: A 51-year-old female was seen for a rapidly enlarging neck mass associated with progressive dyspnea and dysphagia. Despite being a telemedicine visit due to the COVID-19 pandemic, significant enlargement and deformity of the neck were startling. The patient was clinically and biochemically euthyroid with a TSH of 2.49 mIU/L (0.5-5.0 mIU/L) and normal FT4. She denied a personal or family history of thyroid disease and neck irradiation. Thyroid ultrasound revealed a 5.3 cm hypoechoic, wider than tall nodule with smooth margins in the left lobe, and a 1.9 cm hypoechoic and taller than wide nodule in the right lobe of the thyroid. A CT scan of the neck also revealed a 1.2 cm lymph node in the left lateral aspect of the thyroid. FNAB of the nodules showed Hurthle cells in a background of crushed lymphocytes with occasional large atypical lymphoid cells. Flow cytometry performed on a repeat FNAB specimen revealed no evidence of lymphoma. A repeat CT scan of the neck performed 2 weeks later due to worsening symptoms demonstrated middle mediastinal lymphadenopathy and a large 8x4.7x4.7 cm mass contiguous with the thyroid with a 3.8 cm cystic collection within. The patient was scheduled for an oncology consultation due to the high likelihood of lymphoma but was admitted to another institution with symptoms. She underwent a thyroid core biopsy and flow cytometry, which again failed to reveal her diagnosis. She then underwent left partial thyroidectomy, and pathology demonstrated infiltrates of small lymphocytes with histiocytes, interspersed with clusters of large, irregular, multilobate cells consistent with Reed-Sternberg cells. She was diagnosed with nodular sclerosing variant of classical HL. Chemotherapy was instituted with rapid improvement in symptoms. Clinical Lesson: Primary HL of the thyroid is extremely rare and most often presents as a rapidly enlarging neck mass with or without compressive symptoms. Diagnosis with FNAB is challenging and can be misinterpreted as lymphocytic thyroiditis. In our patient, flow cytometry was performed twice and was falsely negative. The treatment of HL differs entirely from that of other primary thyroid cancers and thyroiditis. Hence, in patients presenting with classic symptoms, a high index of suspicion is needed to make an accurate and prompt diagnosis of HL.

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