Utility of Flow Cytometry in the Diagnostic Evaluation of Recurrent or Persistent Nodular Lymphocyte-Predominant Hodgkin Lymphoma

Abstract Objectives We previously reported that lymph nodes involved by nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) had unique flow cytometric features. To further validate their diagnostic utility, we have examined the flow cytometry features in cases of persistent lymphadenopathy (LAD) prior to the diagnosis of NLPHL or recurrent LAD following diagnosis and treatment for NLPHL. Methods We retrospectively identified nine patients (2-3 specimens per patient for a total of 20 specimens) with persistent or recurrent lymphadenopathy before or after they were diagnosed with NLPHL between the ages of 13 and 76 years. Their histopathology diagnoses were reviewed and flow cytometry data were reanalyzed. Results Based on our published criteria (Am J Clin Pathol 2016;145:107-115), positive flow cytometry findings (at least 12% of T cells expressing CD57 or at least 3% of T cells coexpressing CD4 and CD8) were seen in 18 specimens. Based on histopathology, 11 of them were correctly diagnosed as NLPHL, 3 of them initially underdiagnosed as atypical lymphoid proliferation, and 4 of them initially incorrectly diagnosed as negative or progressive transformation of germinal centers (PTGCs). The flow cytometry studies showed similar expression patterns of CD57, CD4, and CD8 in T cells and very similar high percentages of CD57+ T cells and CD4+CD8+ T cells between initial biopsies and subsequent biopsies in these patients. Negative flow cytometry findings were seen in two specimens pathologically confirmed as negative for NLPHL in two patients after treatment. Their initial diagnostic specimens showed positive flow cytometry findings, different from that seen in the subsequent specimens posttreatment. Conclusion Flow cytometry appears to be a useful adjunct in detecting early or relapsed NLPHL, especially in atypical lesions. The presence of positive flow cytometry features is very sensitive in detecting recurrent or persistent NLPHL, while absence of positive flow cytometry features helps rule out NLPHL.

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Flow Cytometry Analysis of Recurrent or Persistent Lymphadenopathy in Patients with Nodular Lymphocyte-Predominant Hodgkin Lymphoma

Clinical Oncology and Research ◽

10.31487/j.cor.2021.08.05 ◽

2021 ◽

pp. 1-6

Author(s):

James Z. Huang ◽

Savanah D. Gisriel ◽

Kristle Haberichter ◽

Sara Huang ◽

James Z. Huang

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Hodgkin Lymphoma ◽

Expression Patterns ◽

Flow Cytometric Analysis ◽

Flow Cytometry Analysis ◽

Flow Cytometric ◽

Multiple Biopsies ◽

Highly Sensitive ◽

Reactive Hyperplasia

Objectives: We recently examined the utility of flow cytometric analysis in the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) by examining reactive T-cell features. This study aims to compare these features in sequential biopsies of persistent or recurrent lymphadenopathy in patients with NLPHL. Methods: We reanalysed the histopathology and flow cytometry findings of 9 patients with multiple biopsies for persistent or recurrent lymphadenopathy and either initial or recurrent NLPHL. A flow cytometry signature was considered suggestive of NLPHL if ≥12% of T-cells expressed CD57 or ≥3% of T-cells co-expressed CD4 and CD8. Results: A flow cytometry signature considered suggestive of NLPHL was seen in 18 of 20 specimens. Based on histopathology, 11 were diagnosed as NLPHL, 3 were initially underdiagnosed as atypical lymphoid proliferation, and 4 were initially incorrectly diagnosed as negative or progressive transformation of germinal centers. Flow cytometry showed similar expression patterns of CD57 and CD4/CD8 in T-cells between initial and subsequent biopsies. The remaining 2 specimens lacked the flow cytometry signature suggestive of NLPHL and were histopathologically diagnosed as reactive hyperplasia. Conclusion: Flow cytometry analysis based on our criteria is highly sensitive in detecting NLPHL. Correlation with the cytospin cytology may increase the diagnostic specificity. A negative flow essentially ruled out the possibility of NHLPHL.

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Flow Cytometric Detection of the Double-Positive (CD4+CD8+)/PD-1bright T-Cell Subset Is Useful in Diagnosing Nodular Lymphocyte-Predominant Hodgkin Lymphoma

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2020-0726-oa ◽

2021 ◽

Author(s):

Zhongchuan Will Chen ◽

Juanita Wizniak ◽

Chuquan Shang ◽

Raymond Lai

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cell ◽

Hodgkin Lymphoma ◽

B Cell Lymphoma ◽

The Other ◽

Non Hodgkin Lymphoma ◽

Flow Cytometric ◽

Large B Cell

Context.— Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is characterized by neoplastic lymphocyte-predominant cells frequently rimmed by CD3+/CD57+/programmed death receptor-1 (PD-1)+ T cells. Because of the rarity of lymphocyte-predominant cells in most cases, flow cytometric studies on NLPHL often fail to show evidence of malignancy. Objective.— To evaluate the diagnostic utility of PD-1 in detecting NLPHL by flow cytometry, in conjunction with the CD4:CD8 ratio and the percentage of T cells doubly positive for CD4 and CD8. Design.— Flow cytometric data obtained from cases of NLPHL (n = 10), classical Hodgkin lymphoma (n = 20), B-cell non-Hodgkin lymphoma (n = 22), T-cell non-Hodgkin lymphoma (n = 5), benign lymphoid lesions (n = 20), angioimmunoblastic T-cell lymphomas (n = 6) and T-cell/histiocyte–rich large B-cell lymphomas (n = 2) were analyzed and compared. Results.— Compared with the other groups, NLPHL showed significantly higher values in the following parameters: CD4:CD8 ratio, percentage of T cells doubly positive for CD4 and CD8, percentage of PD-1–positive T cells, and median fluorescence intensity of PD-1 expression in the doubly positive for CD4 and CD8 subset. Using a scoring system (0–4) based on arbitrary cutoffs for these 4 parameters, all 10 NLPHL cases scored 3 or higher, as compared with only 3 cases from the other groups, producing an overall sensitivity of 100% and a specificity of 96% (72 of 75). Two of the 3 outliers were non-Hodgkin lymphoma, and both showed definitive immunophenotypic abnormalities leading to the correct diagnosis. The remaining outlier was a case of T-cell/histiocyte–rich large B-cell lymphoma. Conclusions.— The inclusion of anti–PD-1 in flow cytometry is useful for detecting NLPHL in fresh tissue samples, most of which would have otherwise been labeled as nondiagnostic or reactive lymphoid processes.

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Flow cytometric detection of perforin upregulation in human CD8 T cells

Cytometry Part A ◽

10.1002/cyto.a.20596 ◽

2008 ◽

Vol 73A (11) ◽

pp. 1050-1057 ◽

Cited By ~ 45

Author(s):

Adam R. Hersperger ◽

George Makedonas ◽

Michael R. Betts

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Flow Cytometric

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Early Response of CD8+ T Cells in COVID-19 Patients

Journal of Personalized Medicine ◽

10.3390/jpm11121291 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1291

Author(s):

Deni Ramljak ◽

Martina Vukoja ◽

Marina Curlin ◽

Katarina Vukojevic ◽

Maja Barbaric ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

T Cells ◽

Mrna Expression ◽

Cd8 T Cells ◽

Expression Profiles ◽

Expression Patterns ◽

Effector Memory ◽

Healthy Controls ◽

Ifn Γ

Healthy and controlled immune response in COVID-19 is crucial for mild forms of the disease. Although CD8+ T cells play important role in this response, there is still a lack of studies showing the gene expression profiles in those cells at the beginning of the disease as potential predictors of more severe forms after the first week. We investigated a proportion of different subpopulations of CD8+ T cells and their gene expression patterns for cytotoxic proteins (perforin-1 (PRF1), granulysin (GNLY), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK)), cytokine interferon-γ (IFN-γ), and apoptotic protein Fas ligand (FASL) in CD8+ T cells from peripheral blood in first weeks of SARS-CoV-2 infection. Sixteen COVID-19 patients and nine healthy controls were included. The absolute counts of total lymphocytes (p = 0.007), CD3+ (p = 0.05), and CD8+ T cells (p = 0.01) in COVID-19 patients were significantly decreased compared to healthy controls. In COVID-19 patients in CD8+ T cell compartment, we observed lower frequency effector memory 1 (EM1) (p = 0.06) and effector memory 4 (EM4) (p < 0.001) CD8+ T cells. Higher mRNA expression of PRF1 (p = 0.05) and lower mRNA expression of FASL (p = 0.05) at the fifth day of the disease were found in COVID-19 patients compared to healthy controls. mRNA expression of PRF1 (p < 0.001) and IFN-γ (p < 0.001) was significantly downregulated in the first week of disease in COVID-19 patients who progressed to moderate and severe forms after the first week, compared to patients with mild symptoms during the entire disease course. GZMK (p < 0.01) and FASL (p < 0.01) mRNA expression was downregulated in all COVID-19 patients compared to healthy controls. Our results can lead to a better understanding of the inappropriate immune response of CD8+ T cells in SARS-CoV2 with the faster progression of the disease.

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CD161 Is Expressed in a Subset of T-Cell Prolymphocytic Leukemia Cases and Is Useful for Disease Follow-up

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqz060 ◽

2019 ◽

Vol 152 (4) ◽

pp. 471-478

Author(s):

Scott R Gilles ◽

Sophia L Yohe ◽

Michael A Linden ◽

Michelle Dolan ◽

Betsy Hirsch ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Nk Cells ◽

Retrospective Review ◽

T Cell Subsets ◽

Prolymphocytic Leukemia ◽

Flow Cytometry Data ◽

Flow Cytometric ◽

Flow Cytometric Evaluation

AbstractObjectivesCD161 (NKRP1) is a lectin-like receptor present on NK cells and rare T-cell subsets. We have observed CD161 expression in some cases of T-cell prolymphocytic leukemia (T-PLL) and found it to be useful in follow-up and detection of disease after treatment.MethodsRetrospective review of T-PLL cases with complete flow cytometry data including CD161.ResultsWe identified 10 cases of T-PLL with flow cytometric evaluation of CD161 available. Six of these cases were positive for CD161 expression. All CD161-positive cases were positive for CD8 with variable CD4 expression, whereas all CD161-negative cases were negative for CD8. In a case with two neoplastic subsets positive and negative for CD8, only the former expressed CD161.ConclusionsThese novel results suggest that CD161 is often aberrantly expressed in a defined subset of T-PLL positive for CD8. We are showing the utility of this immunophenotype in diagnosis and follow-up.

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High frequency of circulating HBcAg-specific CD8 T cells in hepatitis B infection: a flow cytometric analysis

Clinical & Experimental Immunology ◽

10.1046/j.1365-2249.2001.01561.x ◽

2001 ◽

Vol 124 (3) ◽

pp. 435-444 ◽

Cited By ~ 6

Author(s):

S. Matsumura ◽

K. Yamamoto ◽

N. Shimada ◽

N. Okano ◽

R. Okamoto ◽

...

Keyword(s):

T Cells ◽

Hepatitis B ◽

Cd8 T Cells ◽

High Frequency ◽

Flow Cytometric Analysis ◽

Hepatitis B Infection ◽

Flow Cytometric

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Monitoring of T-cell acute lymphoblastic leukemia by flow cytometry

Open Medicine ◽

10.2478/s11536-010-0044-3 ◽

2010 ◽

Vol 5 (6) ◽

pp. 651-658

Author(s):

Miglė Janeliūnienė ◽

Rėda Matuzevičienė ◽

Laimonas Griškevičius ◽

Zita Kučinskienė

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Acute Lymphoblastic Leukemia ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Flow Cytometric ◽

Analysis Strategy ◽

Cell Acute Lymphoblastic Leukemia ◽

Positive Results ◽

Color Marker

AbstractMinimal residual disease (MRD) predicts the outcome of acute lymphoblastic leukemia (ALL). Flow cytometry (FC) is one of the most sensitive and most applicable methods for MRD diagnostics, but there is still no agreement on the “gold standard” of the method. We tried to optimize flow cytometric MRD detection in T-ALL. Fourteen adults and 11 children with T-ALL and 12 normal bone marrow (BM) donors were enrolled in the study. We found that the most common phenotypic aberrations in T-ALL were TdT and CD99 coexpression on T-cells in BM. Therefore for MRD detection we developed a limited four-color marker panel (TdT/CD7/cCD3/CD19 and CD99/CD7/cCD3/CD2) and a standard analysis strategy. This assay was evaluated on BM of healthy controls. Less than 0.01% TdT+ or CD99 bright T-cells were found in normal BM. MRD was detected in 9 adult patients and 1 child at different time-points of treatment. The average TdT and CD99 mean fluorescence intensity (MFI) value of residual blasts fluctuated during therapy, but it still remained higher than MFI of normal T-cells. Our established MRD detection method differentiated leukemic lymphoblasts with sensitivity in the range of 0.01% and did not give any false positive results in normal BM.

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P01.13 MERTK signaling is critical for T cell proliferation and memory

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.26 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A14.2-A15

Author(s):

RM Powell ◽

MJW Peeters ◽

A Rachbech ◽

PT Straten

Keyword(s):

T Cells ◽

Cell Division ◽

T Cell ◽

Cd8 T Cells ◽

Central Memory ◽

T Cell Proliferation ◽

Flow Cytometric ◽

Donor T Cells ◽

Memory Differentiation

BackgroundOverexpression of TAM receptors, including MERTK, in some cancers are integral for chemoresistance, proliferation and metastasis.1 Our group has previously demonstrated that T cells also express MERTK and engagement of MERTK signaling is responsible for increased proliferation, functional capacity and metabolic fitness.2 It is therefore important to further study the effect of MERTK inhibition on T cell function in the context of cancer treatments where MERTK inhibitors may play a role. Here we provide evidence that MERTK inhibition impacts greatly on T cell proliferation, specifically reducing phosphorylated mTOR. We have also demonstrated that MERTK expression is increased on CD8 central memory subsets during longterm expansion providing evidence that this signaling pathway may be important for sustaining T memory responses.Materials and MethodsFlow cytometric analysis was used to investigate the effect of titration of MERTK small molecule inhibitor UNC2025 on healthy donor T cells activated with CD3/CD28 dynabeads. Cell trace dye was used to track proliferation of CD4 and CD8 T cells along with markers of memory differentiation (CCR7 and CD45RO), activation (CD137) and function (IFNy, Tnfa and IL-2). MERTK signaling was assessed using phospho flow cytometric methodology of phosphorylated mTOR, AKT, ERK1/2, p38-MAPK and STAT5. Long term cultures of donor T cells of up to 28 days were investigated for MERTK expression alongside memory differentiation.ResultsWe demonstrated that moderate concentrations of MERTK inhibitor reduced proliferation of activated T cells. Despite inhibition of cell division, cell size still increased 2 fold compared to resting cells and cell viability remained unchanged. Additionally, the proportion of central memory to effector memory populations and intracellular cytokine production was not impacted. Analysis of molecules involved in MERTK signaling revealed that phosphorylated mTOR was significantly modulated following the addition of MERTK inhibitor. Long term culture of CD8 T cells demonstrated MERTK was significantly increased following early and late re-stimulation, and expression of MERTK was strongly associated with central memory subsets.ConclusionsOur results demonstrate that inhibition of MERTK signaling on T cells reduces cell division where mTOR is significantly impacted. Despite this, other functional aspects, such as intracellular cytokine production remain unchanged. Therefore, interruption of MERTK signaling on T cells has a specific effect on cell division rather than cytotoxic function on a cell by cell basis. This has potential ramifications on the use of MERTK inhibitors to treat tumors where the ability to form substantial cytotoxic T cell populations might be reduced. In addition, increased MERTK expression on central memory subsets during long term culture suggests this signaling pathway could be critical for generating memory pools of T cells and provide new avenues for the improvement of adoptive T cell therapy protocols.ReferencesCummings CT, Deryckere D, Earp HS, Graham DK. Molecular pathways: MERTK signaling in cancer. Clin Cancer Res 2013;19(19):5275–5280.Peeters MJW, Dulkeviciute D, Draghi A, et al. MERTK Acts as a Costimulatory Receptor on Human CD8+T Cells. Cancer Immunol Res 2019;7(9):1472–1484.Disclosure InformationR.M. Powell: None. M.J.W. Peeters: None. A. Rachbech: None. P.T. Straten: None.

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3236 Identification of exhaustive markers in cytotoxic T-cells to guide immune modulation in hepatocellular carcinoma ex vivo

Journal of Clinical and Translational Science ◽

10.1017/cts.2019.33 ◽

2019 ◽

Vol 3 (s1) ◽

pp. 13-13

Author(s):

Lauren Norell Krumeich ◽

Tatiana Akimova ◽

Jason Stadanlick ◽

Abhishek Rao ◽

Neil Sullivan ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Cd8 T Cells ◽

Tumor Response ◽

Checkpoint Inhibitors ◽

Ex Vivo ◽

Receptor Expression ◽

Inhibitory Receptors ◽

Surface Receptors ◽

Study Population

OBJECTIVES/SPECIFIC AIMS: Objective: apply checkpoint inhibitors that are specific to the exhaustive markers expressed on tumor CD8+ T-cells ex vivo in order to improve cytokine release and cytotoxic function in comparison to two control groups: (1.) T-cells that receive no antibodies; (2.) T-cells that receive standard inhibition with PD-1 and CTLA-4 antibodies only. Long-term objective: provide personalized medicine in the treatment of HCC by using checkpoint inhibitors that are specific to the receptors expressed by an individual tumor. METHODS/STUDY POPULATION: The study population includes patients undergoing liver transplantation or surgical resection for HCC. Two grams of tumor, two grams of healthy liver tissue at least one centimeter from the tumor margin, and 50 milliliters of blood will be obtained. Solid tissue will be mechanically and enzymatically disrupted and CD8+ T-cells will be isolated from all sites. Using flow cytometry, the expression of surface receptors PD-1, CTLA-4, LAG-3, TIM-3, BTLA, CD244, and CD160 will be categorized in each tissue to identify which receptors are upregulated in the tumor microenvironment. Up to three antibodies specific to the upregulated receptor(s) on the tumor T-cells will be applied per specimen. The experimental arm will receive these antibodies and co-stimulation with CD3/CD28 and will be compared to two controls. One control will receive only CD3/CD28, and the other will receive CD3/CD28 in addition to the standard combination of PD-1 and CTLA-4 inhibitors. From each condition, flow cytometry will be used to assess the mean production of interleukin-2, tumor necrosis factor-α, interferon-γ, granzyme B, and perforin expression as an assessment of T-cell function. RESULTS/ANTICIPATED RESULTS: Preliminary data from the peripheral blood of healthy controls confirms that the developed flow cytometry panels effectively identify the surface receptors and cytokine production of CD8+ T-cells. Two patients have successfully been enrolled in this study. It is predicted that T-cells extracted from the tumor will express more inhibitory receptors than normal liver or peripheral blood and will have increased function after they are targeted with checkpoint inhibitors that are specific to the inhibitory surface receptors they express. DISCUSSION/SIGNIFICANCE OF IMPACT: HCC is the second leading cause of cancer-related death worldwide and therapeutic options are limited for patients who are not surgical candidates. T-cells are a critical component of the anti-tumor response to HCC. However, T-cells can develop an exhausted phenotype characterized by up-regulated inhibitory receptors (PD-1, CTLA-4, LAG-3, TIM-3, CD-244, CD-160, BTLA) and decreased function, allowing for immune escape. Clinical trials using combined checkpoint inhibition with PD-L1 and CTLA-4 antibodies have been considered a breakthrough for patients with advanced HCC, as up to 25% show an objective tumor response. The explanation for the varied susceptibility to checkpoint inhibition remains unknown and is hypothesized to be secondary to inconsistencies in the expression of surface inhibitory receptors. Although inhibitory receptor expression has been shown to be upregulated under conditions of hepatitis and/or HCC, there has been no single study to effectively investigate the expression of all known inhibitors in order to better explore the interplay between them. It will be of great academic interest and clinical purpose to evaluate individual receptor expression and engage the correlating antibodies given the possibility of synergism between receptors and the need for a more profound anti-tumor T-cell response in HCC.

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