Flow cytometry characterization of leukemic phase of nasal NK/T-cell lymphoma in tumor biopsies and peripheral blood

Introduction: Aggressive T cell lymphomas (TCL) are a heterogenous group of lymphomas that are frequently associated with poor outcomes. Autologous stem cell transplantation (ASCT) is recommended according to the NCCN guidelines and by practice standards for most subtypes as a consolidation for patients in first remission. A large prospective study of up-front ASCT by the Nordic Lymphoma group identified age, ECOG performance status <2, and bone marrow involvement as important prognostic factors. We have identified peripheral blood involvement by flow cytometry at diagnosis in up to one third of patients with aggressive TCL and analyzed whether this was a prognostic factor for outcomes after ASCT. Methods: We retrospectively analyzed data from consecutively treated patients (pts) with aggressive T-cell lymphomas who underwent ASCT at our institution from July 2009 to February 2019. Patient and disease characteristics were summarized using descriptive statistics. Kaplan-Meier analysis was used to estimate progression free survival (PFS) that was defined as the time from SCT to the first evidence of recurrence, and overall survival (OS) that was defined as the time from SCT to death or last institutional follow up with a hematologist. We collected data on age, co-morbidities, disease subtype, stage, response to therapy and treatment both pre and post SCT. Flow cytometry was obtained at diagnosis and phenotype of atypical circulating cells was compared with immunophenotype from tumor biopsy specimens. Results: 50 pts with TCL who received ASCT were identified for this analysis. Of this population, 41 (80%) of pts had peripheral blood flow available at the time of initial diagnosis. T-cell lymphoma types included peripheral T cell lymphoma not otherwise specified (PTCL NOS, 17 pts), angioimmunoblastic T cell lymphoma (AITCL, 15pts), ALK negative anaplastic large cell lymphoma (ALCL, 1pt), enteropathy-type T-cell lymphoma (EATL, 2pts), extranodal natural killer T-cell lymphoma (NKTCL, 2pts) and panniculitis like T cell lymphoma (2 pts) (Table 1). Median age of the cohort was 62 years (range 20-75 years) and all patients included had an ECOG PS 0-1 at the time of diagnosis. The majority had stage 4 disease (36/41, 87.8%), but analysis included a small number of patients with stage 2 (1/41, 2.4%) and stage 3 (4/41,9.7%) disease. Bone marrow involvement by morphologic criteria was noted on bone marrow biopsy in 8/41 (19.5%) pts; bone marrow was negative in 28/41 or 61% pts and not evaluated in 8/41 or 19.5% pts. Flow cytometry of peripheral blood performed as part of initial staging was positive for circulating malignant cells in 13/41 pts (31.7%) at the time of diagnosis. All patients underwent ASCT in first remission. The median PFS and OS were 15.2 and 29.9 months respectively in the flow positive group, while neither median PFS nor OS were reached in the flow negative group (Figures 1 and 2). Flow cytometry results from time of diagnosis was not strongly associated with PFS (log rank, p = 0.39), however, it was associated with overall survival (log rank, p = 0.012). There were 11 deaths in the cohort- 4 in the flow negative group and 7 in the flow positive group. Further, when bone marrow involvement was evaluated, 7 of 13 pts with positive flow cytometry (53.8%) and 5 of 28 (17.8%) pts with negative flow cytometry had BM involvement, suggesting a correlation between positive bone marrow and detection of lymphoma cells in the peripheral blood at the time of diagnosis. Conclusions: We demonstrate in our cohort of patients that detection of circulating lymphoma cells at diagnosis by flow cytometry was associated with a worse outcome in patients with aggressive T cell lymphomas undergoing ASCT as a consolidation in first remission. Larger cohorts will be needed to validate these findings, but these results suggest peripheral blood involvement by sensitive flow cytometry may identify patients with worse outcomes who might benefit from a more aggressive strategy such as allogeneic stem cell transplantation or alternative consolidation strategies. Disclosures Huntington: Bayer: Consultancy, Honoraria; Pharmacyclics: Honoraria; Celgene: Consultancy, Research Funding; DTRM Biopharm: Research Funding; Genentech: Consultancy; AbbVie: Consultancy. Isufi:Celgene: Consultancy; Novartis: Consultancy; Astra Zeneca: Consultancy. Foss:Seattle Genetics: Consultancy, Other: fees for non-CME/CE services ; Mallinckrodt: Consultancy; miRagen: Consultancy; Spectrum: Other: fees for non-CME/CE services ; Eisai: Consultancy; Acrotech: Consultancy.

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Comprehensive Flow‐Cytometry‐Based Immunophenotyping Analysis for Accurate Diagnosis and Management of Extranodal NK/T Cell Lymphoma, Nasal Type

Cytometry Part B Clinical Cytometry ◽

10.1002/cyto.b.21838 ◽

2019 ◽

Vol 98 (1) ◽

pp. 28-35

Author(s):

Jian‐Chao Wang ◽

Xue‐Qin Deng ◽

Wei‐Ping Liu ◽

Li‐Min Gao ◽

Wen‐Yan Zhang ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Cell Lymphoma ◽

Accurate Diagnosis ◽

T Cell Lymphoma ◽

Nasal Type ◽

Diagnosis And Management ◽

Nk T Cell

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Immunophenotypic Characterization of CD45RO+ and CD45RA+ T Cell Subsets in Peripheral Blood of Peripheral T Cell Lymphoma Patients

Cell Biochemistry and Biophysics ◽

10.1007/s12013-014-0008-3 ◽

2014 ◽

Vol 70 (2) ◽

pp. 993-997

Author(s):

Wenzhong Yang ◽

Xinyan Jia ◽

Yang Su ◽

Qi Li

Keyword(s):

T Cell ◽

Peripheral Blood ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

T Cell Subsets ◽

Peripheral T Cell Lymphoma

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Diagnostic Value of protocadherin 15 on NK/T Cell Lymphoma

Blood ◽

10.1182/blood.v124.21.5396.5396 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5396-5396

Author(s):

Jiali Zhou ◽

Caigang Xu

Keyword(s):

T Cell ◽

Cell Lines ◽

Peripheral Blood ◽

Cell Lymphoma ◽

Normal Population ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Control Group ◽

Nk T Cell ◽

Experimental Group

Abstract Objective: NK/T cell lymphoma is a unique subtype of non-Hodgkin’s lymphoma, accounting for 5% to 15% of lymphomas, and shows a prediction for the crowd of Asia, central and South America. Nasal, nasopharynx and oropharynx regions are commonly involved. Lesions may also occur in the upper respiratory tract, gastrointestinal tract, testis and other parts. Diagnosis of NK/T cell lymphoma is dependent on the pathological study of lesion biopsy specimens. Nevertheless, poor quantity or quality of specimens, such as tissue extensive necrosis often become an obstacle to the early diagnosis. Therefore, investigating an easy sampling, accurate and stable experimental marker has profound clinical significance for the diagnosis of NK / T-cell lymphoma. Protocadherin15 (PCDH15) is a member of the cadherin family, which encoding gene located on chromosome 10q21-22. Rouget-Quermalet found a secreted isoform PCDH15 in the cracking sediment of human YT-type NK/T-cell lymphoma cell lines 8 years ago. PCDH15 was found not expressed in fresh or activated normal peripheral blood cells, CD4/CD8 positive lymphocytes, NK cells, normal spleen, lymph nodes, tonsils reactivity and CD34 + cord blood cells, only specifically expressed in three kinds of NK cell lymphoma cell lines. PCDH15 was not expressed in the biopsies of diffuse large B-cell lymphoma, mantle cell lymphoma and other types of lymphoma, but specifically expressed in NK/T lymphoma biopsy tissues. Since PCDH15-SI was found in the human NK/T-cell lymphoma cell lines, its relationship with NK / T-cell lymphoma as well as other types of lymphoma was rarely reported, and studies of clinical specimens are still blank. The purpose of this study is to investigate what whether the PCDH15 is specifically expressed in NK/T-cell lymphoma biopsies, and whether the PCDH15-SI, as a secreted isoform, is specifically present in peripheral serum of newly diagnosed NK/T-cell lymphoma or not. Materials and methods: 1. Screening biopsy paraffin blocks of NK/T-cell lymphoma(2010.1～2013.10) and non-NK/T-cell lymphoma(2012.10～2013.11) in West China Hospital of Sichuan University, which have 45cases and 33 cases stained by immunohistochemistry respectively and blank controls were set. 2. Collecting peripheral blood of NK/T-cell lymphoma, non-NK/T-cell lymphoma and normal population in the same Hospita. PCDH15 content of serum samples were detected by enzyme-linked immunosorbent content analysis (ELISA) method. Results: 1. Results of immunohistochemistry showed that PCDH15 were expressed both in the experimental group NK/T-cell lymphoma and the control group with non-NK/T cell lymphoma. High expression rate in the NK/T-cell lymphoma group was higher than in non-NK/T cell lymphoma. The differences of PCDH15 expression levels between the two groups, or among the different anatomical structures were not statistically significant (P > 0.05). 2. PCDH15-SI can be detected in peripheral blood in the normal population, the experimental group of NK/T-cell lymphoma as well as in the control group of non-NK/T-cell lymphoma, which the level of PCDH15-SI in those patients with newly diagnosed disease was higher than that of the normal population and the serum level in the non-NK/T-cell lymphoma patients was higher than that in the NK/T-cell lymphoma patients(P < 0.05). Conclusions: 1. PCDH15 was expressed in biopsies of different types of lymphoma, and the degree of expression in NK/T-cell lymphoma was significantly higher. 2. PCDH15 can’t be used to diagnose NK/T cell lymphoma as a marker. Disclosures No relevant conflicts of interest to declare.

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The predictive value of dynamic monitoring of peripheral blood lymphocyte to monocyte ratio in patients with extranodal NK/T cell lymphoma

Cancer Cell International ◽

10.1186/s12935-019-0993-9 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Shengnan Zhang ◽

Mengjuan Li ◽

Fangfang Yuan ◽

Lin Chen ◽

Ruihua Mi ◽

...

Keyword(s):

T Cell ◽

Treatment Response ◽

Peripheral Blood Lymphocyte ◽

Peripheral Blood ◽

Blood Lymphocyte ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Dynamic Monitoring ◽

Lymphocyte To Monocyte Ratio ◽

Nk T Cell

Abstract Background To investigate the value of dynamic monitoring peripheral blood lymphocyte-to-monocyte (LMR) ratio in evaluating the treatment response and prognosis of patients with extranodal NK/T cell lymphoma (ENKTL). Methods A total of 148 patients with ENKTL were retrospectively analyzed in the Affiliated Tumor Hospital of Zhengzhou University between March 2012 and March 2018. The optimal cut-off value of LMR was determined using the receiver operating characteristic curve (ROC) method, then patients were divided into low LMR group and high LMR group. The LMR level was dynamically measured at various time points, and the relationships between LMR and therapeutic response, and survival were analyzed. Results The complete remission rate (CR) was 85.7% in patients with high LMR at diagnosis, which was remarkably higher than that of patients with low LMR at diagnosis (64.9%) (P = 0.009). The 5-year overall survival (OS) and progression-free survival (PFS) were 49.28% and 44.89% in the low LMR group, respectively; 5-year OS and PFS in the high LMR group were 84.50% and 67.12%, respectively, significantly longer (P values were < 0.001 and 0.034, respectively). The OS and PFS of patients with elevated LMR after treatment were longer than those with decreased LMR after treatment (all P values < 0.05). The LMRs at relapse were significantly lower in both high and low LMR groups than those of the last follow-up (P values were 0.001 and 0.016, respectively). Univariate and multivariate analysis demonstrated that low LMR was an independent risk factor for poor prognosis in ENKTL patients (P values were < 0.001 and 0.009, respectively). Conclusions Lymphocyte to monocyte ratio can be used as an indicator of treatment response, prognosis and recurrence in patients with ENKTL. Low LMR before and after treatment is a poor prognostic factor.

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Flow Cytometric Characterization of 129 Cases of Peripheral T Cell Lymphoma Not Otherwise Specified (PTCL NOS) and Angioimmunoblastic T Cell Lymphoma (AITL)

Blood ◽

10.1182/blood.v126.23.2667.2667 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2667-2667

Author(s):

Greg Hapgood ◽

Anja Mottok ◽

Graham W Slack ◽

Randy D. Gascoyne ◽

Christian Steidl ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cell Lymphoma ◽

Cell Suspensions ◽

T Cell Lymphoma ◽

Peripheral T Cell Lymphoma ◽

Flow Cytometric ◽

Not Otherwise Specified

Abstract Background: Peripheral T cell lymphoma not otherwise specified (PTCL NOS) and angioimmunoblastic T cell lymphoma (AITL) together comprise approximately half of all peripheral T cell lymphomas. Malignant T cells in AITL and in a subset of PTCL-NOS exhibit a phenotype mimicking that of normal T-follicular helper (TFH) cells. Immunohistochemical (IHC) studies performed on paraffin-embedded tissues are a mainstay of diagnostic histopathology, but can be difficult to interpret when the malignant T cells show limited cytological atypia and when there are abundant infiltrating reactive T cells. Flow cytometry represents an alternate means to define the cellular immunophenotype, but requires access to single cell suspensions of viable tumor cells. Flow cytometry has certain additional benefits over IHC including highly quantitative measurement of multiple antigens simultaneously and statistical power afforded by analyzing tens of thousands of individual cells. We report here immunophenotypic characterization of a large cohort of cases of PTCL NOS and AITL using a 12-color flow cytometry assay and correlation of immunophenotypic features with clinical outcomes. Methods: Cases of PTCL-NOS and AITL spanning a 24 year period (1990-2014) for which viably frozen cell suspensions from diagnostic lymph node biopsies were available were identified within the British Columbia Cancer Agency (BCCA) lymphoma database. Cryopreserved cell suspensions were thawed and stained with a 12-color panel including 11fluorochrome-conjugated antibodies against lineage (CD45, CD19, CD3, CD4, CD8), pan-T cell (CD2, CD5, CD7), and TFH cell (CD10, CD279, CXCR5) markers, plus DAPI for gating of live cells. Flow cytometric data was acquired on a Becton Dickinson FACSAria3 instrument as part of a sorting experiment to isolate tumor cell subpopulations. Data was analyzed by conventional gating and bivariate plot display using FlowJo software and correlated with clinical outcome data. Results: 74 cases of PTCL-NOS and 55 cases of AITL were analyzed. The median age at diagnosis was 57 years (y) for PTCL NOS (male:female 1.6) and 75 y for AITL (male:female 1.0). The median follow up for living patients was 5.15 y. The median specimen viability was 36.5% (range 0.8-89.3%) and median specimen tumour content was 64.3% of viable events (range 0.98-91.8%). Aberrant T cell immunophenotypes were identified in 50 of 74 cases (68%) of PTCL NOS and 36 of 55 cases (65%) of AITL. Five specimens had more than one identifiable immunophenotypically aberrant T cell population. For the 50 PTCL NOS cases with an aberrant immunophenotype, 31 (62%) demonstrated loss of CD3 and 42 (84%) demonstrated loss of CD7. About half of cases were CD4+CD8- (27, or 54%) including 11 (22%) that exhibited a TFH-like phenotype (positive for at least 2 of the 3 assayed TFH markers), while the remaining were CD4-CD8- (23, or 46%). TFH-like cells were also identified in 11 of 24 (46%) cases lacking an aberrant T cell immunophenotype. For the 36 AITL cases with an aberrant immunophenotype, 21 (58%) demonstrated loss of CD3 and 29 (80%) demonstrated loss of CD7. The majority of cases were CD4+ (30, or 83%) including 21 (58%) that exhibited a TFH-like phenotype, while the remaining were either CD8+ (4, or 11%) or CD4-CD8- (2, or 6%). TFH-like cells were also identified in 7 of 19 (37%) cases lacking an aberrant T cell immunophenotype. Similar to other patient cohorts, the 5 y PFS and 5 y OS was 21% and 40%, respectively, for PTCL NOS and 17% and 28%, respectively, for AITL. The presence of an aberrant phenotype, CD3 status, and CD4/CD8 status were not associated with prognosis in either PTCL subtype. A preliminary analysis suggests loss of CD7 expression in PTCL NOS is associated with an inferior outcome. Analysis of archival material and exploration in a validation cohort is ongoing. Discussion: An aberrant population of varying abundance was detected in >65% of specimens for PTCL NOS and AITL. The aberrant immunophenotype in PTCL NOS was evenly split between CD4+CD8- and CD4-CD8- cases. Interestingly, nearly half of CD4+ cases showed evidence of TFH-like differentiation, possibly corresponding to the TFH-like variant of PTCL NOS. The aberrant immunophenotype in AITL was typically CD4+ and often with co-expression of TFH-associated markers. Loss of CD7 and CD3 were the most common abnormalities. Loss of CD7 may demonstrate a poor-risk group of patients with inferior outcomes in PTCL NOS. Disclosures Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding.

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