Neutropenia Associated With T-Cell Large Granular Lymphocyte Leukemia: Long-Term Response to Cyclosporine Therapy Despite Persistence of Abnormal Cells

Abstract T-cell large granular lymphocyte (T-LGL) leukemia is clinically indolent, but is associated with severe neutropenia in approximately 50% of cases. The pathogenesis of the neutropenia is unclear. We report reversal of severe neutropenia associated with T-LGL leukemia in five patients treated with cyclosporine (CSA). All five had persistent neutrophil counts below 0.5 × 109/L, two had agranulocytosis, and four had recurrent infections. Increased populations of LGL were present in blood and marrow, with a T-LGL immunophenotype (CD3+CD8+CD16±CD56±CD57+) shown by multiparameter flow cytometry, and clonal T-cell receptor (TCR) gene rearrangements in two of two pretreatment blood samples studied. CSA was initiated at doses of 1 to 1.5 mg/kg orally every 12 hours, with subsequent dose adjustments based on trough serum levels. Four patients attained normal neutrophil counts with CSA alone; one required addition of low-dose granulocyte-macrophage colony-stimulating factor. Time to attainment of 1.5 × 109/L neutrophils ranged from 21 to 75 days. Attempts to taper and withdraw CSA resulted in recurrent neutropenia. Three patients have maintained normal neutrophil counts on continued CSA therapy for 2, 8, and 8.5 years. Two patients died 1.7 and 4.6 years after initiation of CSA despite normal neutrophil counts—one of metastatic melanoma and one of complications after aortofemoral bypass surgery. Despite resolution of neutropenia, increased populations of T-LGL cells have persisted in all patients during CSA therapy, as shown by morphology and flow cytometry and by the presence of clonal TCR gene rearrangements in four patients' posttreatment blood samples. We conclude that CSA is an effective therapy for neutropenia associated with T-LGL leukemia, and that resolution of neutropenia despite persistence of abnormal cells implies that CSA may inhibit T-LGL secretion of yet unidentified mediators of neutropenia.

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Neutropenia Associated With T-Cell Large Granular Lymphocyte Leukemia: Long-Term Response to Cyclosporine Therapy Despite Persistence of Abnormal Cells

Blood ◽

10.1182/blood.v91.9.3372.3372_3372_3378 ◽

1998 ◽

Vol 91 (9) ◽

pp. 3372-3378 ◽

Cited By ~ 2

Author(s):

Raman Sood ◽

Carleton C. Stewart ◽

Peter D. Aplan ◽

Hiroyuki Murai ◽

Pamela Ward ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Large Granular Lymphocyte ◽

Severe Neutropenia ◽

Multiparameter Flow Cytometry ◽

Gene Rearrangements ◽

Aortofemoral Bypass ◽

Blood Samples ◽

Abnormal Cells ◽

Lgl Leukemia

T-cell large granular lymphocyte (T-LGL) leukemia is clinically indolent, but is associated with severe neutropenia in approximately 50% of cases. The pathogenesis of the neutropenia is unclear. We report reversal of severe neutropenia associated with T-LGL leukemia in five patients treated with cyclosporine (CSA). All five had persistent neutrophil counts below 0.5 × 109/L, two had agranulocytosis, and four had recurrent infections. Increased populations of LGL were present in blood and marrow, with a T-LGL immunophenotype (CD3+CD8+CD16±CD56±CD57+) shown by multiparameter flow cytometry, and clonal T-cell receptor (TCR) gene rearrangements in two of two pretreatment blood samples studied. CSA was initiated at doses of 1 to 1.5 mg/kg orally every 12 hours, with subsequent dose adjustments based on trough serum levels. Four patients attained normal neutrophil counts with CSA alone; one required addition of low-dose granulocyte-macrophage colony-stimulating factor. Time to attainment of 1.5 × 109/L neutrophils ranged from 21 to 75 days. Attempts to taper and withdraw CSA resulted in recurrent neutropenia. Three patients have maintained normal neutrophil counts on continued CSA therapy for 2, 8, and 8.5 years. Two patients died 1.7 and 4.6 years after initiation of CSA despite normal neutrophil counts—one of metastatic melanoma and one of complications after aortofemoral bypass surgery. Despite resolution of neutropenia, increased populations of T-LGL cells have persisted in all patients during CSA therapy, as shown by morphology and flow cytometry and by the presence of clonal TCR gene rearrangements in four patients' posttreatment blood samples. We conclude that CSA is an effective therapy for neutropenia associated with T-LGL leukemia, and that resolution of neutropenia despite persistence of abnormal cells implies that CSA may inhibit T-LGL secretion of yet unidentified mediators of neutropenia.

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Long-Term Outcome of Cyclosporin A Therapy for T-Cell Large Granular Lymphocyte Leukemia.

Blood ◽

10.1182/blood.v110.11.2063.2063 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2063-2063

Author(s):

Attaphol Pawarode ◽

Paul K. Wallace ◽

Laurie A. Ford ◽

Maurice Barcos ◽

Maria R. Baer

Keyword(s):

Platelet Count ◽

T Cell ◽

Growth Factor ◽

Cyclosporin A ◽

Gene Rearrangement ◽

Large Granular Lymphocyte ◽

Severe Neutropenia ◽

Lgl Leukemia

Abstract T-cell large granular lymphocyte (T-LGL) leukemia is a rare indolent lymphoproliferative disorder diagnosed by presence of >2,000/μL peripheral CD3+CD8+CD16+/−CD56 +/−CD57+ LGL and/or evidence of T-LGL clonality by T-cell receptor (TCR) gene rearrangement, accompanied by neutropenia and/or other cytopenias. We reported the efficacy of cyclosporin A (CsA) in the treatment of LGL-associated neutropenia [Blood1998;91:3372–8] and we report here long-term follow-up of our larger patient (pt) cohort. Between 1982 and 2006, 18 pts were diagnosed with T-LGL leukemia with the above strict criteria (M:F 11:7; median age, 67; range, 48–84 years). Mean follow-up from diagnosis is 59 (range, 2–303) months. Autoimmune phenomena were present in 6 (33%), including pure red cell aplasia in two, lymphocytic colitis and ascites in one, and erosive osteoarthritis and pulmonary granulomatous vasculitis in one other. Splenomegaly was present in 6 (33%). Median (range) hemoglobin (Hb), absolute neutrophil count (ANC) and platelet count were 10.4 (3.6–15.5) g/dL, 1,100 (0–5,900)/μL and 152,000 (69,000–583,000)/μL. Anemia, neutropenia (ANC<1,000/μL), severe neutropenia (ANC<500/μL) and thrombocytopenia were present in 9 (50%), 13 (72%), 6 (33%), and 5 (28%), respectively. Four (22%) had one cytopenia, ten (56%) bicytopenia and one (6%) pancytopenia. Median (range) absolute number and percentage of peripheral LGL by flow cytometry were 1,147 (59–9,327)/μL and 31 (3.3–66.9) %. TCR gene rearrangement was present in 16 (89%). Fourteen pts (78%) required treatment early; median treatment-free survival was only 1.0 (95% CI, 0–8.4) months. Indications for initiation of treatment included Hb<9 g/dL, ANC<500/μL, ANC<1,000/μL with recurrent infections and platelet count<50,000/μL. CsA was initiated in 11 pts (61%). The median duration of CsA therapy was 47 (range, 4.5–186) months. Four pts were still receiving CsA at last follow-up. Four pts (40%) achieved sustained complete hematologic improvement (HI) by MD Anderson response criteria [Leuk Res 2007;31:939–45], 1 (10%) major HI and 2 minor HI (20%), resulting in an overall HI of 70%. One other (10%) had mixed HI and 2 (20%) had progressive disease. G-CSF was initiated simultaneously with, and added later to, CsA in 1 (10%) and 2 (20%) pts, respectively, and erythropoietin (Epo) in 1 (10%) and 3 (30%), respectively. The addition of growth factor resolved neutropenia and/or anemia in two additional pts, resulting in overall and complete HI of 80% and 50% respectively. Median time (range) to maximum HI with CsA, G-CSF and Epo were 2.5 (0.2–27), 0.7(0.6–1.9) and 1.5 (0.9–1.9) months, respectively. Renal insufficiency, hypertension and/or hypomagnesemia occurred in 7 (70%). CsA tapering and discontinuation were successful in two (40%) of the five pts who achieved complete HI, 91 and 186 months after initiation. Four pts (22.2%) have not required any treatment 8, 8, 33 and 80 months after diagnosis, and two received Epo and one HIV therapy alone. All except one were alive at the mean follow-up time of 59 (range, 2–303) months and median survival has not been reached. Thus T-LGL leukemia usually requires treatment early after diagnosis, but has an excellent prognosis. CsA with or without growth factor support is an effective treatment, leading to overall and complete HI in 80% and 50%, respectively. Successful tapering and discontinuation of therapy may be possible in 40% of pts who achieve long-term complete HI.

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Clonotype Switching Indicates Propensity for Clonal Outgrowth From Diverse Components of the T Cell Repertoire In T Cell Large Granular Lymphocyte Leukemia.

Blood ◽

10.1182/blood.v116.21.1171.1171 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1171-1171

Author(s):

Michael J. Clemente ◽

Marcin W. Wlodarski ◽

Aaron D. Viny ◽

Mohammed Shaik ◽

Nelli Bejanyan ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Autoimmune Disease ◽

Significant Proportion ◽

Surrogate Marker ◽

The Body ◽

Large Granular Lymphocyte ◽

Large Granular Lymphocyte Leukemia ◽

Flow Cytometric ◽

Lgl Leukemia

Abstract Abstract 1171 T cell large granular lymphocyte leukemia (T-LGLL) is characterized by the chronic proliferation of cytotoxic T lymphocytes (CTL) and is often associated with lineage-restricted cytopenias, autoimmune disease and B cell dyscrasias. In many patients clinical features of a true leukemia are absent. Lack of transformation, absence of progressive increase in blood counts, and a paucity of recurrent chromosomal defects or mutations clearly distinguish LGL from typical leukemic development. Thus, some forms of T-LGLL resemble more of a reactive process and possibly represent an extreme pole of oligo- or polyclonal immune reactions to viruses, autoantigens, or perhaps even exaggerated immune tumor surveillance. We have shown that a highly skewed T cell receptor (TCR) variable beta (Vβ) repertoire is strongly associated with monoclonality of TCR CDR3 regions by sequencing and therefore, detection of a Vβ expansion by flow cytometric clonotyping can serve as a surrogate marker for the presence of a clonal CTL process. Flow cytometric Vβ typing offers an opportunity to study the dynamics of the CTL clonal progression during therapy and throughout the clinical course of disease. We studied 124 patients who not only met the WHO guidelines for the diagnosis of T-LGLL but also show skewing of the Vβ spectrum by flow cytometry consistent with the mono/oligoclonal process persistent for over 6 months; 100% of cases demonstrated both TCRγ rearrangement and abnormal CTL population by flow cytometry. LGL count >900/μL was present in 73% of patients (mean 2513±3571 cells/μL). A pathologic clonal Vβ expansion was defined as > mean + 2SD of controls (n=65). Expansions identified by the Vβ panel were present in 92% (mean clone size 55±28%) and 2% had a borderline expansion (within 20% of 2SD) and 3% a δ/γ CD8+ TCR expansion. In 6% the Vβ expanded clone expressed CD4. Absolute clone count (ACC) was calculated by the Vβ contribution multiplied by the absolute CD8 (or CD4) count per μL of blood. ACC correlated well with LGL count (p<.0001, R2=.58). Clinically, patients presented with anemia (25%), neutropenia (9%), pancytopenia (19%), thrombocytopenia (3%), or multi-lineage cytopenia (29%), and 15% of patients were asymptomatic. In order to assess clonal kinetics, 62 of these patients were available for serial Vβ measurements with a follow up range of 0.5–8 years. Paired statistical analysis of clonal dynamics pre and post therapy (cyclophosphamide, alemtuzumab, methotrexate or cyclosporine) demonstrated a significant decrease in ACC between responders and refractory patients (p=.024). Unexpectedly, some patients displayed a change in the dominant clone as demonstrated by a switch in the major clonal Vβ T cell population, i.e. “clonotype switch.” Overall, 32% exhibited a clonotype switch during the study period, while others exhibited the persistence of multiple clones (22%); in 46% the initially diagnosed Vβ monoclonal expansion persisted. Those with multiple clones were more likely to change clonal dominance (p=.05). Clonotype switch was observed in both in relapsed and in refractory patients, and also was frequently accompanied by a change in clinical hematologic features. Significant absolute lymphocytosis (>4000 lymphocytes/μL) was present in 34/124 (27%) patients while 66 had normal lymphocyte counts and 24 were lymphopenic. Of the patients followed serially who clonotype switched, only 3/20 (15%) had absolute lymphocytosis, suggesting that 2 distinct subtypes of T-LGLL may exist. Patients with high lymphocyte and LGL counts may represent a true leukemic process, are less likely to have associated autoimmune disease or second malignancy, and their dominant clone is stable, suggestive of a single transformed precursor. In contrast, in patients with clonal CTL expansions not associated with absolute lymphocytosis, clonotype switching is difficult to reconcile with a true autonomous leukemic process. In sum, our results suggest that in a significant proportion of patients with T-LGL leukemia, the propensity for clonal CTL dominance is not inherent to an aberrant molecular event within the abnormal CTL clone but may rather be related to extrinsic chronic antigenic drive and immune dysregulation. Furthermore, our results lend credence to the body of evidence suggesting that T-LGL leukemia may not, in many instances, be a true leukemia, and may best be classified as T-LGL lymphoproliferation. Disclosures: No relevant conflicts of interest to declare.

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A Multiplex, Droplet Digital PCR Assay for the Detection of T-Cell Receptor Excision Circles and Kappa-Deleting Recombination Excision Circles

Clinical Chemistry ◽

10.1373/clinchem.2019.308171 ◽

2019 ◽

Vol 66 (1) ◽

pp. 229-238 ◽

Cited By ~ 1

Author(s):

Tracie Profaizer ◽

Patricia Slev

Keyword(s):

Flow Cytometry ◽

T Cell ◽

T Cell Receptor ◽

Reference Gene ◽

Cell Receptor ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Blood Samples ◽

Healthy Donors ◽

Excision Circles

Abstract BACKGROUND T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count <20 copies/μL and 17.7% had a KREC count <20 copies/μL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.

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Detection of human T-cell leukemia/lymphoma virus, type II, in a patient with large granular lymphocyte leukemia

Blood ◽

10.1182/blood.v80.5.1116.1116 ◽

1992 ◽

Vol 80 (5) ◽

pp. 1116-1119 ◽

Cited By ~ 95

Author(s):

TP Jr Loughran ◽

T Coyle ◽

MP Sherman ◽

G Starkebaum ◽

GD Ehrlich ◽

...

Keyword(s):

T Cell ◽

Large Granular Lymphocyte ◽

Enzyme Linked Immunosorbent Assay ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Enzymatic Amplification ◽

Htlv Infection ◽

Human T Cell ◽

Polymerase Chain ◽

Lgl Leukemia

Abstract We studied a patient with large granular lymphocyte (LGL) leukemia for evidence of human T-cell leukemia/lymphoma virus (HTLV) infection. Serum from this patient was positive for HTLV-I/II antibodies by enzyme- linked immunosorbent assay (ELISA) and was confirmed positive in Western blot and radioimmunoprecipitation assays. Results of a synthetic peptide-based ELISA showed that the seropositivity was caused by HTLV-II and not HTLV-I infection. Analyses of enzymatic amplification of DNA from bone marrow sections using the polymerase chain reaction (PCR) were positive for HTLV-II specific gag, pol, env, and pX gene sequences. Cloning and sequencing of amplified products showed that the HTLV-II pol and pX sequences in patient DNA differed from the sequences of 17 other HTLV-II isolates examined in our laboratory. HTLV infection may have a role in some patients in the pathogenesis of LGL leukemia.

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Lack of gene rearrangement and mRNA expression of the beta chain of the T cell receptor in spontaneous rat large granular lymphocyte leukemia lines.

Journal of Experimental Medicine ◽

10.1084/jem.161.5.1249 ◽

1985 ◽

Vol 161 (5) ◽

pp. 1249-1254 ◽

Cited By ~ 36

Author(s):

C W Reynolds ◽

M Bonyhadi ◽

R B Herberman ◽

H A Young ◽

S M Hedrick

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Antigen Receptor ◽

Cell Receptor ◽

Large Granular Lymphocyte ◽

Beta Chain ◽

T Cell Antigen Receptor ◽

Cell Antigen Receptor ◽

Cell Antigen ◽

Lgl Leukemia

Using the murine cDNA clone for the beta chain of the T cell antigen receptor, we have examined four highly cytotoxic rat large granular lymphocyte (LGL) leukemia lines for the expression of unique rearrangements and mRNA transcription of the genes coding for the T cell antigen receptor. In contrast to normal rat T cells and nine rat T cell lines, the LGL leukemia lines exhibited no detectable gene rearrangements in the beta chain locus after digestion of LGL DNA by four restriction enzymes. Northern blots containing RNA from these LGL tumor lines demonstrated a low level of aberrant or nonrearranged beta chain transcription (less than 10 copies per cell) but virtually no translatable 1.3 kilobase message. These results demonstrate that LGL leukemia lines which mediate both natural killer (NK) and antibody-dependent cell-mediated cytotoxicity (ADCC) activities do not express the beta chain of the T cell receptor. The nature of the NK cell receptor for antigen remains elusive.

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Clonal drift demonstrates unexpected dynamics of the T-cell repertoire in T-large granular lymphocyte leukemia

Blood ◽

10.1182/blood-2011-02-338517 ◽

2011 ◽

Vol 118 (16) ◽

pp. 4384-4393 ◽

Cited By ~ 34

Author(s):

Michael J. Clemente ◽

Marcin W. Wlodarski ◽

Hideki Makishima ◽

Aaron D. Viny ◽

Isabell Bretschneider ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Cell Receptor ◽

Large Granular Lymphocyte ◽

T Cell Repertoire ◽

Cell Repertoire ◽

Large Granular Lymphocyte Leukemia ◽

Tcr Repertoire ◽

Serial Measurements ◽

Β Chain

AbstractT-cell large granular lymphocyte leukemia (T-LGLL) is characterized by chronic lymphoproliferation of cytotoxic T lymphocytes (CTLs) and is associated with lineage-restricted cytopenias. Introduction of T-cell receptor (TCR) variable β-chain (Vβ) monoclonal antibodies has facilitated identification and enumeration of clonal CTLs by flow cytometry. A highly skewed TCR Vβ repertoire identified by flow cytometry is strongly associated with monoclonal CDR3 regions by quantitative sequencing and positive TCRγ rearrangement assays. Therefore, Vβ expansions can serve as surrogate markers of CTL clonality to assess clonal kinetics in T-LGLL. We analyzed the TCR repertoire in 143 patients, 71 of which were available for serial measurements over 6 to 96 months. Although the majority (38/71, 54%) maintained a consistent monoclonal expansion, many (26/71, 37%) unexpectedly displayed a change in the dominant clone, whereby the original CTL clone contracted and another emerged as demonstrated by Vβ typing. Our results demonstrate that the T-cell repertoire is more dynamic in T-LGLL than recognized previously, illustrating the heterogeneity of disorders under this categorization.

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10 A multiparameter flow cytometry assay to monitor natural killer cell proliferation and activation in immuno-oncology clinical trials

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0010 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A10-A10

Author(s):

Jennifer Tsau ◽

Brittney Atzmiller ◽

David Quinn ◽

Tanya Mulvey ◽

Sema Kurtulus ◽

...

Keyword(s):

Clinical Trial ◽

Flow Cytometry ◽

Cell Proliferation ◽

T Cell ◽

Natural Killer ◽

Nk Cell ◽

Multiparameter Flow Cytometry ◽

Parent Population ◽

Flow Cytometry Assay ◽

Cd Markers

BackgroundNatural Killer (NK) cells have garnered increasing interest as potential cellular therapies or as targets of biotherapeutic agents due to their ability to kill tumor cells in a non-antigen dependent manner. Hence, measurement of NK cell proliferation and/or activation following treatment can serve as a useful biomarker for assessing the efficacy of immunomodulatory therapies.MethodsWe developed a novel 13-parameter flow cytometry panel incorporating cell differentiation (CD) markers important for identification of NK cell subsets (CD56, CD16), their proliferation (Ki-67), activation (CD25, CD335, NKG2D) and inhibition (CD159a) status. Additionally, CD markers that identify other cellular subsets known to be amenable to cytokine modulation (e.g., CD3 and CD14) were included for concurrent monitoring of T cell proliferation and monocyte activation. Method validation focused on analytical sensitivity, specificity and precision as key criteria of assay performance using peripheral blood mononuclear cells (PBMCs) stimulated with NK cell-activating cytokines and resting PBMCs from healthy donors.ResultsThe assay design allowed for robust quantitation of NK cell, T cell and monocyte functionalities. Lower limit of quantification (LLOQ) of target biomarker population was determined to be 1.0% of the parent population, based upon an analysis of 110 key target populations that displayed a co-efficient of variation (CV) of ≤25% and their frequencies ranged from 0.1% to 97.8% of the parent population. Additionally, ≤25% CV was observed in precision assessments, confirming the repeatability and reproducibility of the assay. Clinical trial utility of the assay was verified on cryopreserved PBMCs from patients with a variety of solid tumor malignancies. In these patients, the assay could clearly identify proliferating and activated NK cells, as well as proliferating T cells and activated monocytes, thus demonstrating its suitability for clinical trial applications.ConclusionsWe developed and validated a novel multiparameter flow cytometry assay that allows for simultaneous measurement of proliferation, activation and inhibitory status of key immune cell subsets. Thus, this assay can help shed light on the mode of efficacy of novel therapeutic agents that modulate the immune system, aimed at treatment of cancer and autoimmune diseases.

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Polarized CTL Responses Detected in Patients with Autoimmune Neutropenia.

Blood ◽

10.1182/blood.v104.11.1459.1459 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1459-1459

Author(s):

Marcin W. Wlodarski ◽

Yadira Narvaez ◽

Alexander Rodriquez ◽

Jaroslaw P. Maciejewski

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Significant Proportion ◽

Diagnostic Algorithm ◽

Diagnostic Methods ◽

Cytotoxic T Cell ◽

Autoimmune Neutropenia ◽

Pcr Cloning ◽

Myeloid Progenitor Cells ◽

Lgl Leukemia

Abstract Drugs and intrinsic bone marrow diseases can explain most of the cases of neutropenia, and true autoimmune neutropenia (AIN) is a diagnosis of exclusion. Anti-neutrophil antibodies are not reliable, and their absence does not preclude the diagnosis of AIN. Lineage-restricted cytopenias, including neutropenia, were associated with T cell Large Granular Lymphocyte leukemia (T-LGL), but the diagnosis of this condition involves positive TCR rearrangement and flow cytometric identification of a pathologic cytotoxic T cell (CTL) population. These routinely applied methods have a limited sensitivity and rely on the presence of a high frequency of clonal cells in the sample. AIN, similar to T-LGL, may be related to a CTL-mediated process. We hypothesize that AIN, in a portion of patients, is a CTL-mediated disease in which myeloid progenitor cells are the targets. Consequently, in those patients, polarized expansions of CTL clones may be detected if efficient and sensitive diagnostic methods will be applied. Previously, we developed a diagnostic algorithm for the identification and quantification of clonal expansions in T-LGL based on the molecular analysis of TCR- utilization pattern. We studied a cohort of patients with various degrees of neutropenia (N=23) that was unexplained by clinical grounds and standard laboratory testing. Anti-neutrophil antibodies were found in 6 of these patients; in 3 patients, serum-mediated inhibition (>20%) of colony formation by normal hematopoietic progenitor was found, but there was no correlation between antibodies and serum inhibition. For detection of CTL expansions in AIN, VB typing and VB specific RT-PCR were applied followed by PCR cloning and sequencing of a large number of clones, and determination of expanded CDR3 clonotypes. When no expansion was detected by flow cytometry, multiplex PCR was used to amplify the whole VB spectrum. If identical CDR3 regions were detected by sequencing of at least 22 clones, CDR3 fragments of appropriate VB families were subcloned and sequenced, and immunodominant (identical clones occurring repetitively) were identified. Using this approach, we found only 2 expanded clones in 24 healthy donors. Those expanded clones accounted for 20% of a given VB family, or 0.7% of the CD8+ repertoire (as calculated by multiplication of clonal expansion within VB family by VB family contribution to the whole CTL population). In AIN we found expansion in 9 of 21 patients (3 of them were not detected by VB flow cytometry). Clonal frequency was 40%± 13% of a given VB family or 13%± 14% of the total CD8+ population. The presence of expanded CTL did not correlate with anti-neutrophil antibodies of serum-mediated colony inhibition. By comparison, CTL clones found in patients with T-LGL leukemia (N=75) comprised 68% of a given VB family, or 43% of the entire VB repertoire. We conclude that, using sensitive approaches, CTL expansions can be detected in a significant proportion of patients with AIN. These cases may represent minor variants of an autoimmune process that operates in T-LGL leukemia. The antigens that trigger these expansions likely may be shared. Clinically, detection of the CTL-mediated process in neutropenia may point toward rational immunosuppressive therapy aimed at T cells.

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Large Granular Lymphocyte Leukemia: Clonality Reconsidered.

Blood ◽

10.1182/blood.v110.11.3102.3102 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3102-3102

Author(s):

Wesley Witteles ◽

Bing Zhang ◽

Iris Schrijver ◽

Daniel Arber ◽

Jason Gotlib ◽

...

Keyword(s):

Capillary Electrophoresis ◽

T Cell ◽

High Resolution ◽

Clinical Characteristics ◽

Cell Receptor ◽

Concordance Rate ◽

Large Granular Lymphocyte ◽

Third Generation ◽

Primer Sets ◽

Lgl Leukemia

Abstract Background: T-cell large granular lymphocyte (LGL) leukemia is widely considered to represent a monoclonal proliferation of lymphocytes. Clonality assessment methods have evolved from Southern blots (first-generation) to polymerase chain reaction with heteroduplex electrophoresis (second-generation) to high-resolution capillary electrophoresis (third-generation) testing. Aims: To determine if third-generation T-cell clonality assays result in a higher frequency of oligoclonal results, to compare the concordance for testing at the T-cell receptor (TCR) gamma (TCRG) and TCR beta (TCRB) loci, and to compare the clinical characteristics of patients with monoclonal vs. oligoclonal TCRs. Methods: The study population consisted of patients from August 1999-April 2007 with elevated circulating LGLs and cytopenia(s). TCRG locus clonality was determined by both the heteroduplex method and capillary electrophoresis in 35 patients. 89 samples were tested for TCRG and TCRB clonality using the Biomed II PCR primer sets and capillary electrophoresis on an ABI 3100 automated DNA sequencer. Determinations of clonality were made independently by three pathologists blinded to the clinical characteristics of the patients. Results: A total of 93 patients (median age 50 years, 53% female) were evaluated. Median absolute neutrophil count was 1.56 × 109/L (range 0.2–7.8 × 109/L), median lymphocyte count was 1.81 × 109/L (range 0.6–13 × 109/L), and median hemoglobin was 13 g/dL (range 6.3–17.4 g/dL). The concordance rate for TCRG clonality testing by the heteroduplex and capillary electrophoresis methods was only 40%. The primary difference was a striking increase in the frequency of oligoclonal results by the capillary electrophoresis method (p= 0.00007). All of these samples appeared monoclonal by the lower resolution heteroduplex assay (Table 1). Concordance for clonality for TCRG vs. TCRB was 54% (Table 2). All samples had monoclonality or oligoclonality demonstrated at TCRG or TCRB, but only 26% were monoclonal at both loci. The clinical characteristics for the 23 patients with monoclonal TCRG and TCRB appeared similar to the 23 patients with oligoclonal TCRG and TCRB. The median age in both groups was 53 years, with 61% of patients in each group requiring treatment after a median of 36.8 and 38.6 months of follow-up, respectively. Discussion: The high resolution of capillary electrophoresis appears to result in a much greater proportion of oligoclonal TCRG results, which by the older heteroduplex method would have been considered monoclonal. Furthermore, the concordance rate at TCRG and TCRB appears to be remarkably low. Though oligoclonal T-cell populations are generally believed to be transient and reactive processes, the clinical characteristics of our oligoclonal and monoclonal cohorts did not differ significantly. Conclusion: Capillary electrophoresis frequently identifies patients with oligoclonal TCR whose clinical features are indistinguishable from those of patients with classic monoclonal LGL leukemia. Heteroduplex Monoclonal Negative Oligoclonal Total Monoclonal 12 1 1 14 Capillary Electrophoresis Negative 4 2 0 6 Oligoclonal 15 0 0 15 Total 31 3 1 35 TCRG Monoclonal Negative Oligoclonal Total Monoclonal 23 3 12 38 TCRB Negative 7 0 1 8 Oligoclonal 15 3 25 43 Total 45 6 38 89

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