Chronic Lymphoproliferative Disorders Research Articles

Detection of Apoptotic Lymphocytes Through Sysmex XN-1000 As a Diagnostic Marker for Mononucleosis Syndrome.

The infectious mononucleosis (IM) includes clue elements, apoptotic and atypical lymphocytes. In IM, the evaluation of dot plot provided by Sysmex XN-1000 analyzer revealed a stretched lymphocytic cluster, white cell differential channel (WDF), on cytogram. In this study, we analyzed 698 samples that include 39 IM, 76 chronic lymphoproliferative disorders, 25 nonclonal lymphocytosis, and 40 healthy donors. Five hundred eighteen samples with other diseases or interference were evaluated. The algorithm was validated on 40,000 files that were received from internal database of Sysmex-Dasit. The analysis of flow cytometry standard (FCS) files in WDF channel and presumed apoptotic lymphocytes counts on side scatter/forward scatter (SSC/FSC) and SSC/SFL (where SFL is side fluorescence) dot plot revealed excellent correlation among apoptotic cells on peripheral blood smear (R(2) =0.79 and 0.75). There was a variation of positional parameters in lymphocyte clusters WX, WY, and WZ. If WX-SSC>500 and WY-SFL>1,000 and WZ-FSC>700, specificity equals to 99% and sensitivity equals to 100%. If nucleated red blood cell (NRBC) <0.03×10(3) /μl, specificity equals to 100%. In received files, positives were 1% adding the simultaneous presence of a percentage of events in the two gates relating to the apoptotic lymphocytes of 1.2% of WBC. On Sysmex XN-1000, dot-plot observation allowed immediate detection of IM. Meanwhile, an algorithm based on the parameters on these plots can be calculated with excellent performance.

Cell size variations of large granular lymphocyte leukemia: Implication of a small cell subtype of granular lymphocyte leukemia with STAT3 mutations

Large granular lymphocyte leukemia (LGL-L) has been morphologically defined as a group of lymphoproliferative disorders, including T-cell large granular lymphocytic leukemia (T-LGL-L), chronic lymphoproliferative disorders of NK cells (CLPD-NK) and aggressive NK cell leukemia. We investigated the morphological features of LGL leukemic cells in 26 LGL-L patients in order to elucidate relationships with current classifications and molecular backgrounds. LGL-L cells were mostly indistinguishable from normal LGL. Patients with STAT3 SH2 domain mutations showed significantly smaller cells compared with patients without STAT3 mutations. Four patients with T-LGL-L showed smaller granular lymphocytes with a median diameter of less than 13μm, which were rarely seen in normal subjects. This small subtype of T-LGL-L was recognized among rather young patients and was associated with D661Y mutations in the STAT3 gene SH2 domain. In addition, all of them showed anemia including two cases with pure red cell aplasia. These results suggest the heterogeneity of T-LGL-L and a specific subtype with small variants of T-LGL-L.

Mature T- and NK-cell non-Hodgkin lymphoma in children and young adolescents.

Mature T/Natural killer (NK)-cell neoplasms of children and the young adolescent population exhibit higher prevalence in Central and South American and Asian populations and many are associated with Epstein-Barr virus (EBV). They are represented in large part by extranodal T/NK cell lymphomas- nasal-type or extra nasal-type, chronic lymphoproliferative disorders of T/NK cells or chronic active EBV disease, systemic EBV-positive lymphoproliferative disorders of childhood, hydroa vacciniforme-like lymphoma, hepatosplenic T-cell lymphoma and primary cutaneous gamma/delta T-cell lymphoma among others. Many T/NK cell neoplasms in this age group are derived from cells of the innate immune system, in contrast to adults where they are predominantly from the adaptive immune system. The genetic basis of T/NK cell lymphomas in children and young adolescents remains largely unknown. Anthracycline-based regimens and haematopoietic stem cell transplants (allogeneic and autologous) are current treatment modalities, however it is anticipated that novel targeted therapeutic agents will be available in the near future.

Large Granular Lymphocytic Leukemia and JAK/STAT Signaling Pathway--Review

Large granular lymphocytic leukemia (LGLL) is a rare lymphoproliferative disorder of clonal expansion of cytotoxic T- or NK-cells in blood and bone marrow, and often associated with autoimmune disorders. According to the current WHO classification of the hematopoietic and lymphoid tissue tumors, the clonal LGL expansions are further classified as T-cell large granular lymphocytic leukemia (T-LGLL), chronic lymphoproliferative disorders of NK cells (CLPD-NK) and aggressive NK cell leukemia. Since there is a general lack of awareness of this disease, some patients may be misdiagnosed or some cases may be missed when diagnosis was done. At present, the pathogenesis of LGLL remains incomplete and unclear, and the therapeutic effects are unsatisfactory. For this reason, it is necessary to find prognostic marks and therapeutic targets of this disease. The constitutive activation of JAK/STAT pathway has been claimed to be involved in the development of LGLL. Recently, the somatic mutations in the SH2 domain of STAT3 in LGLL are frequently observed, which lead to the activation of JAK/STAT pathway. STAT3 is the first molecular markers that are highly specific for LGLL, and STAT3 mutations have been rarely detected in other tumor types studied, thus the STAT3 mutations can be used as molecular markers for LGLL diagnosis and can provide a novel therapeutic target for patients with LGLL.

Low expression of CD200 predicts shorter time-to-treatment in chronic lymphocytic leukemia.

CD200, formerly known as OX-2, is a type I glycoprotein that is expressed on a variety of cell types. CD200 has been shown to be overexpressed in chronic lymphocytic leukemia (CLL). Although previous studies have confirmed the diagnostic value of CD200 in differentiating CLL from to other B-cell chronic lymphoproliferative disorders especially mantle cell lymphoma, whether CD200 has prognostic significance in CLL remains to be determined. We evaluated the mean fluorescence intensity (MFI) of CD200 in 307 consecutive, untreated patients with CLL in our center using flow cytometry. Using a CD200 MFI cutoff of 189.5, these cases could be divided into two groups. Patients with lower CD200 MFI (< 189.5) had a significantly shorter time-to-treatment (TTT) than those with higher CD200 MFI (≥ 189.5) (median TTT: 2 months vs 28 months, p = 0.0008). However, the effect of CD200 MFI on overall survival was not significant (CD200 MFI < 189.5: undefined vs CD200 MFI ≥ 189.5: undefined, P = 0.2379). In subgroup analysis, CD200 MFI retained its prognostic value in patients with favourable characteristics such as Binet stage A disease, mutated IGHV status, normal TP53 or negative CD38 expression. In conclusion, our study identified CD200 MFI as a potential prognostic factor in CLL.

EVOLUŢIA PARTICULARĂ A SINDROAMELOR LIMFOPROLIFERATIVE CRONICE ASOCIATE CU INFECŢIA CU VIRUS HEPATITIC B ŞI REACTIVAREA VIRALĂ – O PROBLEMĂ IMPORTANTĂ CU ABORDARE MULTIDISCIPLINARĂ

Background. The association of hepatitis B virus (HBV) infection with chronic lymphoproliferative disorders becomes a matter of debate, with multidisciplinary approach due to possible viral reactivation after immuno-chemotherapy. Material and methods. We studied two patient groups with chronic lymphoproliferative disorders, one with HBV infection (HbsAg+ and HbsAg- HbcAb+ HBs+/-) – 43 patients and one without viral infection (104 patients). Clinical and paraclinical parameters, therapy, survival of both groups were compared; the occurrence of viral reactivation was followed. Data were statistically analyzed. Results and discussion. The HBV infection group included HbsAg+-ve patients (72%) and occult HBV infection patients. The median onset age for lymphoproliferative disorders HBV+-ve was significantly younger (55 vs. 61 years, p&lt;0.05). Hepatomegaly and abdominal adenopathies were more frequent in HBV patients (p=0.003, respectively p=0.027). Hepatic function was altered in HBV patients both at disease onset (AST p=0.0213, GGT p=0.0002) and after first line therapy (AST p=0.0003, ALT p=0.019, FA p=0.008, GGT p=0.000, total bilirubine p=0.043, INR p=0.0003 and albumine p=0.05). The risk of hepatic dysfunction after first line therapy is increased 6 times by HBV infection (p=0.0009, OR=6.000, 95%CI: 1.9121-18.8272), which could negatively impact the hematological disease evolution by lowering therapeutic dose, leading to an inferior treatment result. Viral reactivation occurred both in HbsAg+-ve (21.42%) and HbsAg- HbcAb+-ve patients (60%). None of the occult carriers received antiviral prophylaxis. Whithin our occult carrier group, the viral reactivation percentage is high, therefore we would reccomand antiviral prophylaxis. The overall survival was similar in both group of patients. Conclusions. HBV-positive chronic lymphoproliferative disorders could become a new pathological entity, with individual clinical features and outcome and possibly with negative impact on long term survival. Antiviral prophylaxis for occult carriers could significantly reduce the viral reactivation rate and therefore the therapeutic guidelines should be revised.

Successful Treatment of Macrophage Activation Syndrome in Chronic Lymphoproliferative Disorder of Natural Killer Cells with Cyclophosphamide

Introduction:Chronic natural killer (NK) cell lymphoproliferative disorders comprise a rare subgroup of diseases characterized by proliferation of NK cells with expression of CD3-/CD16+ or CD56+ on flow cytometry. Clinically, these patients present with cytopenias as well as fatigue and B symptoms. The diagnosis can be challenging due to frequent lack of a unique clonal marker. Chronic NK cell lymphoproliferative disorder can be differentiated from NK cell leukemia based on the absence of chronic EBV infection. Macrophage activation syndrome (MAS) can occur in the setting of hematologic malignancies. Due to the rarity of this diagnosis, there are no randomized controlled trials to determine the optimal therapy. We report the case of an elderly gentleman whom we diagnosed with chronic NK cell lymphoproliferative disorder, complicated by transfusion-dependent autoimmune hemolytic anemia, and MAS, and who was successfully treated with oral cyclophosphamide.Case Report:In March 2014, a 75 year old Caucasian male, who was otherwise in good health, had a syncopal episode. Two months earlier, he had reported dyspnea on exertion and cough. He was diagnosed with bronchitis and these symptoms resolved with a course of antibiotics. Cardiac work-up was unremarkable. Complete blood count demonstrated white blood count of 16.8K with 84% lymphocytes, with hemoglobin of 7.8g/dL and platelets of 264K. Lactate dehydrogenase (LDH) was elevated at 615 IU/L, haptoglobin was undetectable, total bilirubin was mildly elevated at 1.5mg/dL and a Coomb's assay was negative. The patient had adequate iron stores with 50% saturation and erythropoietin level was 76.7ug/L. CT of the abdomen/pelvis demonstrated mild splenomegaly of 14cm. CT scan also revealed numerous thoracic-spine soft lesions. Bone marrow biopsy revealed a hypercellular marrow with appropriate trilineage hematopoiesis, erythroid hyperplasia and increased lymphocytes which were CD2+ CD7+ CD16+ and CD 56 negative cells as well as negative for T and B cell markers. This lymphocyte population was identified as NK cells and comprised 16% of his cells. The patient's transfusion dependence did not decrease despite oral prednisone. He transferred to our university medical center. Repeat flow cytometry after one month on prednisone demonstrated an increase to 70% of the lymphocytes. Further, there was a high suspicion for macrophage activation syndrome given his ferritin of 7,358ng/mL, triglycerides of 290mg/dL and interleukin-2 receptor of 7,250pg/mL. There was no evidence of active hemophagocytosis on the bone marrow biopsy. He continued on prednisone 60mg daily, and started on cyclophosphamide 50mg daily. His clinical course over the next several months was complicated by recurrent fevers, night sweats, mental status changes, and hyponatremia. Sepsis was ruled out as a cause of his fevers and his mental status changes were attributed to be secondary to hyponatremia and MAS. Work-up of his hyponatremia revealed syndrome of inappropriate antidiuretic hormone secretion (SIADH). After 2 months of treatment, his sodium level and mental status normalized. His cyclophosphamide was increased to 100mg daily and maintained at that dose. He was then tapered off prednisone. This patient's B symptoms of fevers and night sweats abated after several months of this therapy, and his transfusion dependence and underlying MAS resolved. The patient has been maintained on cyclophosphamide 100mg for 11 months and has shown resolution of his lymphocytosis and normalization of his blood counts.Discussion:Chronic lymphoproliferative disorders of NK cells continue to be a difficult group of diseases to recognize. Moreover, this case was further complicated by MAS based on elevated interleukine-2 receptor, triglycerides and ferritin levels in the setting of B-symptoms and liver enzyme elevations. This constellation of factors has not been previously described in the literature. Due to lack of clonal markers in NK cells, the diagnosis is frequently made by exclusion. The patient has done very well on oral cyclophosphamide and prednisone alone. We present this case to increase provider awareness and hopefully allow for improved diagnosis and treatment options in the future. DisclosuresNo relevant conflicts of interest to declare.

Utility of CD200 Expression and CD20 Antibody Binding Capacity in Differentiating Chronic Lymphocytic Leukemia from Other Chronic Lymphoproliferative Disorders

Introduction: B-chronic lymphoproliferative disorders (CLPD) are a heterogeneous group of clonal proliferations of mature B lymphoid cells with variable clinical presentations and outcomes. Therefore, accurate classification is crucial for deciding therapy. At present flow cytometry immunophenotyping is a useful tool for diagnosing these diseases. However overlapping immunophenotypes do exist. Recently few studies have highlighted the differential expression of CD200 in various CLPDs. Studies have also shown that quantitation of CD20 antibody binding capacity (ABC) varies in different CLPDs.In this study we therefore, planned to evaluate the utility of CD200 expression and CD20 quantitation by flow cytometry in categorizing various CLPDs and to analyse whether CD200 expression and quantitation of CD20 can be used to differentiate various CLPDs.Methods: A total of 77 patients were recruited in the study. CD200 expression was evaluated semi-quantitatively by comparing with the isotype control; and designated as 1+, 2 + and 3+, when there was <1 log, 1-2 log and >2 log shift in mean fluorescence intensity (MFI) compared with isotype control respectively.For CD20 ABC quantitation, 4 sets of precalibrated quantibrite beads and quantibrite CD20 phycoerythrin antibody (Becton Dickinson, San Jose) were used. ABC was calculated using geometric means on a statistical spread sheet.Results: Out of the 77 patients, 54 (70.1%) were of chronic lymphocytic leukemia (CLL), it being the most common type of CLPD in our group of patients. Other types of CLPDs in our study included 6 (7.8%) patients of mantle cell lymphoma (MCL); 5 (6.5%) of hairy cell leukemia (HCL); 3 (3.9%) each of diffuse large B-cell lymphoma (DLBCL) and unclassified B-cell lymphomas; 2 (2.6%) each of follicular lymphoma (FL) and splenic marginal zone lymphoma (SMZL); and 1 each of small lymphocytic lymphoma (SLL) and lymphoplasmacytic lymphoma (LPL).CD200 expression was seen in 100% CLL cases with a homogenous bright (2+) intensity. On the contrary CD200 was uniformly negative in all MCL cases except in 1, however, the intensity was dim (1+). The difference of CD200 expression between CLL and MCL was found to be statistically significant (p<0.001). All cases of HCL and 1 case of FL were also positive for CD200. Other CLPD did not express CD200.Analysis of CD20 ABC showed that CD20 ABC of the lymphoid cells differ among various subtypes and it was significantly lowest in CLL and highest in HCL both on peripheral blood and bone marrow samples. The highest mean CD20 ABC of HCL in comparison to the lowest mean CD20 ABC of CLL was statistically significant (p<0.001). However, there was no statistically significant differences among the CD20 ABC of LPL, MCL, DLBCL, SMZL, SLL and B cell lymphomas unclassified (p>0.05). CD20 ABC of lymphoid cells was also performed in 36 normal controls. A higher CD20 ABC compared to normal controls was seen in HCL and LPL cases only. CLL and other remaining CLPD cases showed CD20 ABC lower than normal controls. Statistical analysis showed that the higher CD20 ABC of normal B lymphocytes in comparison to CLL lymphocytes was statistically significant (p=0.001).Conclusion: In this study, we found that CD200 expression and CD20 quantitation aided in differentiating various CLPDs, especially CLL from MCL. All cases of CLL showed CD200 positivity and low CD20 ABC, compared to MCL where CD200 was predominantly negative and CD20 ABC was high.In addition, amongst all CLPD, HCL had a distinct characteristic pattern for CD200 expression and CD20 ABC, being brightest and highest respectively.Our results indicate that CD200 can be added to routine CLPD panel and is particularly useful in differentiating CLL from MCL.Although, CD20 ABC is useful in differentiating CLPD, adding it to routine panel does not seem practical and cost effective; however, it may be tested in difficult diagnostic cases or where anti-CD20 therapy is planned. DisclosuresNo relevant conflicts of interest to declare.

A Pyrazolo[3,4-d]Pyrimidine Compound Reduces Fyn Phosphorylation and Induces Apoptosis in Large Granular Lymphocyte Leukemia Cells

IntroductionAccording to WHO classification, large granular lymphocyte leukemia (LGL leukemia) includes three entities: T-cell LGL leukemia, chronic NK lymphoproliferative disorders (NK-CLPD) and aggressive NK cell leukemia. Since no standard therapies for immature NK cell neoplasms have been established so far and the overall outcomes are dismal, new therapeutic options are needed.A pyrazolo[3,4-d]pyrimidine library of compounds have already been studied in a previous work by Tintori et al. It has been demonstrated that one of these compounds proved to be efficient against some member of Src-family of nonreceptor protein-tyrosine kinases, in particular against Fyn. Its abnormal activity has been shown to be related to various hematological malignancies.In this context we verified the effect of the pyrazolo[3,4-d]pyrimidine 4c compound on LGL leukemia cells and we investigated its mechanism of action.MethodsThe pyrazolo[3,4-d]pyrimidine 4c compound was tested in a time course MTS test (24, 48, 72 h) at different concentrations (0.1, 0.5, 1, 2, 4, 8, 10 µM) on NK leukemia cells (KHYG1 and NK92), hepatosplenic gamma-delta T cell lymphoma cells (Derl-2 and Derl-7), acute T cell leukemia cells (Jurkat), chronic myeloid leukemia cells (Jurl-MK1), acute myeloid leukemia cells (OCI), multiple myeloma cells (SKM-M1), cervical cancer cells (HeLa) and healthy stem cells (CD34+).Fyn mRNA and protein expression was evaluated in all cell lines by quantitative RT-PCR and western blotting (WB). Fyn mRNA levels were evaluated in cells from 10 healthy controls, 10 T-LGL leukemia, 5 GD-LGL leukemia and 8 NK-CLPD patients by qRT-PCR.Apoptosis and cell cycle studies were performed on KHYG1 after treatment with this compound at 4 µM for 24 h using cytometric analysis by labeling cells with annexin V and propidium iodide. Apoptosis pathway was investigated using proteome profiler human apoptosis array kit; pro-caspase 3, activated caspase 3, Akt and p70S6 kinase were validated by WB. Fyn immunoprecipitation was conducted on treated KHYG1 at 4 µM for 24 h.ResultsThe pyrazolo[3,4-d]pyrimidine 4c compound reduced cell viability in KHYG1 cells (50% of reduction after 24 h of treatment at 4 µM), in Derl-2 cells (about 50% after 72 h at 4 µM), in Derl-7 (about 40% after 72 h at 8 µM), in Jurkat cells (50% after 72 h at 2 µM), in NK92 cells (40% after 72 h at 4 µM). No differences in cell viability were observed in Jurl-MK1, OCI, SKM-M1, HeLa and CD34+. To evaluate Fyn expression level, we performed qRT-PCR and WB showing that both transcript and protein levels were highest in NK and T cells (KHYG1>NK92>Jurkat>Derl-7>Derl-2) compared to other cells. Furthermore, we investigated the viability reduction in treated KHYG1 cells demonstrating that pyrazolo[3,4-d]pyrimidine 4c compound induced 45% of apoptosis caspase-3 mediated and cell cycle arrest in G2/M phase. In these cells we detected a reduction of Fyn phosphorylation after treatment and we also observed a decrease of total levels of Akt and p70S6 kinase, other two protein involved in apoptotic process and cell proliferation.Interestingly, qRT-PCR data showed a significant over-expression of Fyn mRNA level also in T-LGL, GD-LGL and NK-CLPD patients compared to healthy controls (p<0.001).ConclusionIn this study we demonstrated a higher Fyn expression in LGL leukemia patients compared to healthy controls. We also demonstrated an apoptotic effect of pyrazolo[3,4-d]pyrimidine 4c compound on NK leukemia cells and we provided in vitro preliminary evidences that Fyn is its possible cellular target. DisclosuresMusto:Novartis: Honoraria; Celgene: Honoraria; Sanofi: Consultancy; Genzyme: Consultancy; Sandoz: Consultancy; Janssen: Honoraria; Mundipharma: Honoraria; Roche: Honoraria.

The Prognostic Significance of 13q Deletion with Different Size in Patients with B-CLPD

Background: Deletion of 13q14 is the most frequent abnormality in chronic lymphocytic leukemia (CLL). Deletion at of 13q with vary sizes have been described, documenting different involved genes with different deletion area. But the prognostic significance of different deletion size is not well explored and this interesting phenomenon has not been described in other B-cell chronic lymphoproliferative disorders (B-CLPD).Patients and Methods: Interphase fluorescence in situ hybridization (FISH) using locus-specific probes (RB1, D13S25) was performed to detect 13q14 deletions on 344 untreated cases with B-CLPD, including 304 CLL patients and 166 other B-CLPD patients. Different 13q14 deletion area was delineated by RB1 and D13S25.Results: (a) Totally, 145 of 304 CLL patients (47.9%) had 13q14 deletion, which was significantly higher than it in other B-CLPD (25/167, 15.0%; p=.000). 77 of 304 CLL patients (25.3%) and 14 of 166 other B-CLPD patients (8.4%) had RB1 deletion. RB1 deletion always accompanied with D13S25 deletion in both CLL and other B-CLPD, which indicated large area deletion in 13q14. But some patients had only D13S25 deletion without RB1 deletion, indicating small area deletion in 13q14. Of the 145 CLL cases with 13q14 deletion, 73(50.3%) had small deletion and 72 (49.7%) with large deletion. The incidence of small and large deletion in 21 other B-CLPD with 13q14 deletion were in 12(48.0%) and 113(52.0%) patients respectively.(b) In CLL cases, the time to first therapy in the cases with 13q14 deletion was 25 months longer than 16 months in the cases without 13q14 deletion (P=0.035). The progression-free survival (PFS) in cases with 13q14 deletion was also significantly longer (P=0.023) than those without the aberration. In other B-CLPD cases, the PFS and OS in cases with 13q14 deletion was significantly shorter (P=0.030) than those without the aberration. Moreover, large deletions (from RB1 to D13S25) were associated with a significantly shorter (p< 0.05) TFT as well as overall survival (57.5 vs. 135.0 months, p=.075)compared with small deletions.Conclusions: The deletion of 13q can influence clinical outcome of BCLPD and vary in size both in CLL and in other BCLPDs. The deletion size displayed as a poor prognostic factor in other BCLPD cases, which implies the usage of both 13q probes is proposed. DisclosuresNo relevant conflicts of interest to declare.

FTY720 (Fingolimod) Targets the Sphingolipid Pathway and Induces Autophagy-Related Apoptosis in Human Natural Killer Large Granular Lymphocyte Leukemia

Introduction: Natural killer large granular lymphocytic leukemia (NK-LGL) and chronic lymphoproliferative disorder of NK cells (CLPD-NK) are rare disorders of cytotoxic CD3-/CD56+ natural killer cells. Aggressive NK-LGL leukemia patients present with a malignant clinical course and a fatal outcome with median survival time of two months from diagnosis. Aggressive NK-LGL is refractory to conventional chemotherapy and pathogenetic mechanisms remain undefined.The 'sphingolipid rheostat' has been identified as a key player in determining cell fate. The balance between pro-apoptotic ceramide and pro-survival sphingosine-1-phosphate (S1P) in this rheostat is determined by the expression of acid ceramidase (AC) which converts ceramide to sphingosine, and sphingosine kinase-1 and -2 (SPHK1 and SPHK2), which convert sphingosine to S1P. The S1P pathway is involved in cancer pathogenesis, cell survival, and resistance to chemotherapy and radiation, therefore it is considered a potential target for anti-cancer therapy. S1P signals both extracellularly through a family of five related GPCRs; the S1P receptors (S1PR1-5) and intracellularly through various incompletely defined mechanisms. FTY720 is a sphingolipid modulator that targets the S1P system and has potent immunosuppressive and anti-cancer properties. We have previously shown that FTY720 shows therapeutic efficacy in a rat model of NK-LGL leukemia, but its mechanism-of-action in inducing cell death is incompletely defined. The aim of this study is to explore these molecular mechanisms and further characterize the actions of FTY720 in NK-LGL leukemia.Results: We identified that the key enzymes in S1P biosynthesis, SPHK1 and AC, and the S1P receptor S1PR5 were all expressed in LGL cell lines and primary PBMCs from NK-LGL patients. Increased mRNA levels of AC, SPHK1 and S1PR5 were detected in NK-LGL samples (n=8) compared to PBMCs from healthy donors (n=8) (p < 0.05). These results were verified by immunoblot in NK-LGL samples (n=6) versus PBMCs from healthy donors (n=3) (p < 0.05). In vitro treatment of human and rat LGL cell lines (NKL, RNK16 and TL-1) resulted in time- and dose-dependent decreases in cell proliferation and increased apoptosis (IC50 5.0 - 12.5 μM). Interestingly, treatment of the LGL cell lines with the phosphorylated form of FTY720, FTY720-P, which acts antagonistically at the S1PRs, resulted in minimal effects on proliferation and apoptosis. This suggests that FTY720-induced cell death is independent of its effects on S1PRs. However, treatment of LGL cell lines with FTY720 did target multiple sphingolipid pathway members. We observed dose-dependent decreases in AC and S1PR5 protein expression. Interestingly, the combination of FTY720 with LCL204, an AC inhibitor, resulted in additive effects on cell proliferation and apoptosis in the NKL cells.Further characterization of cell death mechanisms demonstrated involvement of autophagy-related apoptosis. We observed increased PARP and caspase-3 cleavage after FTY720 treatment, which occurred after increased expression of LC3A/B and the conversion of LC3-I to LC3-II, suggesting autophagy is being induced prior to apoptosis induction. We identified the mTOR pathway as a target of FTY720. The mTOR pathway negatively regulates autophagy and its inhibition can induce autophagy. FTY720 treatment suppressed mTOR signaling in LGL cell lines as demonstrated by decreased phosphorylation of mTOR, p70S6K and S6 Kinase. These effects preceded the autophagy-related changes in LC3A/B. Furthermore, PARP and caspase-3 cleavage could be blocked by pre-treatment with 3-methyladenine and BafilomycinA1, both inhibitors of autophagy. Interestingly, the effects of FTY720 in LGL cell lines could also be blocked by pre-treatment with autophagy inhibitors, further suggesting that autophagy is required for the FTY720-induced effects. Importantly, these key findings were also reproduced in PBMCs from NK-LGL patients (n=3).Conclusions: Taken together, our findings demonstrate that FTY720 targets multiple sphingolipid pathway members in NK-LGL leukemia and that FTY720-induced cell death is autophagy-dependent. The down-stream targeting of mTOR by FTY720, combined with mTOR's prominent role in inhibiting autophagy, suggests that co-treatment with specific mTOR inhibitors in combination with FTY720 may be a promising and novel therapeutic strategy in LGL leukemia. DisclosuresNo relevant conflicts of interest to declare.

Phenotypic Heterogeneity of Chronic Lymphoproliferative Disorder of NK Cells

BackgroundNK cells represent a subset of lymphocytes belonging to the innate immunity branch typically expressing CD16 and CD56 associated to CD3 negativity. Two major subtypes of NK cells can be distinguished through CD16 and CD56 expression: CD56high/CD16dim/neg NK cells with low cytotoxic function and CD56dim/CD16high NK cells with high cytotoxic function. Recently a subtype of NK cells with memory properties characterized by CD56dim/CD16high/CD57+/CD62L- phenotype has been discovered. Chronic Lymphoproliferative Disorders of Granular Lymphocytes are characterized by the clonal expansion of Large Granular Lymphocyte (LGL) that can be CD3 positive (T-LGLL) or CD3 negative (Chronic Lymphoproliferative Disorder of NK Cell, CLPD-NK). The disease generally has indolent course but some patients develop cytopenia, particularly neutropenia, exposing to potentially lethal bacterial infections. Furthermore, NK-CLPD is usually referred as a more indolent disorder with respect to T-LGLL, with lower incidence of cytopenia and treatment need. CLPD-NK therapy does not differ from that of T-LGLL and is usually represented by an immunosuppressive therapy with low dose cyclophosphamide or methotrexate, with cyclosporine A usually being reserved to refractory patients. Somatic STAT3 mutations represent a new diagnostic marker of these disorders, initially reported in T-LGLL in about 40% oh patients, but also present in CLPD-NK in about 30% of cases.Using flow analysis, the aim of the present study was to identify a subset of CLPD NK patients characterized by a more severe disease requiring a shorter follow-up as compared to patients with a more indolent disease.MethodsIn a cohort of 16 patients affected by CLPD-NK, NK cells were analysed by flow for CD3, CD16, CD56, CD57 and CD62L antigen expression. These patients were studied for the presence of cytopenia and treatment requirement. STAT3 mutation analysis of exon 21 was performed with Sanger sequencing. Finally, p-STAT3 tyr 705 level and total STAT3 level were examined by western blotting.ResultsIn relation to CD16 and CD56 expression, three major NK cells populations can be recognized in CLPD-NK patients: CD56high/CD16neg NK cells, CD56dim/CD16neg NK cells and CD56neg/dim/CD16high. As a consequence, patients can be separated into three groups characterized by the preferentially expansion of one of these populations: 2/16 (13%) with CD56high/CD16neg NK population, 4/16 (25%) with CD56dim/CD16neg NK population and 10/16 (62%) with CD56neg/dim/CD16high NK population. Furthermore, patients with predominance of this last NK cells subset were studied for CD57 and CD62L expression to identify NK cytotoxic subset (CD57-/CD62Llow/neg) and NK memory subset (CD57+/CD62Llow/neg); a NK cytotoxic/memory ratio (C/M ratio) was then calculated. 4 of 10 CD56neg/dim/CD16high patients (40%) were characterized by prevalence of NK cytotoxic cells expansion and high C/M ratio (≥3) while the remaining 6/10 patients were characterized by NK memory cells expansion with low C/M ratio (≤1.6).We then evaluated the presence of cytopenia, in particular neutropenia, in our patients' cohort. Neutropenia was shown in 7/16 (44%) patients with 4/16 (25%) experiencing severe neutropenia. Anemia and thrombocytopenia were less frequent (19% and 6% respectively). Interestingly, 6 out of 7 (86%) neutropenic patients were in the CD56neg/dim/CD16high subset and all patients with severe neutropenia belonged to the high C/M ratio subset. Interestingly, 3 out of 4 patients (75%) of this subset required therapy during the natural history of the disease. Concerning STAT3 mutation analysis, no one mutated patient was found in this setting. By western blot analysis, patients with high C/M ratio presented higher p-STAT3 levels than other patients and normal NK cells.SummaryAlthough CLPD-NK represents an extreme heterogeneous disorder, discrete subtypes of disease characterized by different NK cells population expansion can be identified by flow analysis. Interestingly, this splitting allows to identify a subset of patients with prevalence of CD56neg/dim/CD16high NK cells with high C/M ratio that are characterized by high level of p-STAT3, high frequency of severe neutropenia and treatment requirement. DisclosuresNo relevant conflicts of interest to declare.

Differential Diagnosis of Lymphoid Enhancer-Binding Factor 1 Between Chronic Lymphocytic Leukemia and Other B-Cell Chronic Lymphoproliferative Disorders

Abstract Background : LEF1, lymphoid enhancer factor 1, is a key transcription factor of Wnt signaling pathways. In normal blood cells, the LEF1 mainly expresses in pre-B and T cells, involves in proliferation and differentiation. With mature of the cells, the expression of LEF1 reduces gradually. Its abnormal expression is associated with a variety of tumors, such as various kinds of leukemias and lymphomas. Previous studies have explored the relationship between LEF1 gene and tumorogenesis as well as tumor development, and there is little report on the diagnostic significance of LEF1. Recent studies have shown that the LEF1 protein expression in chronic lymphocytic leukemia (CLL) is significantly higher than other B-cell chronic lymphoproliferative disorders (B-CLPD) and normal controls. Our study aimed to compare the LEF1 gene mRNA expression and the LEF1 protein expression between different subtypes of patients with B-CLPD, then established the differential diagnostic value of LEF1 in B-CLPD. Methods: We determined the expression level of LEF1 protein by immunohistochemistry in bone marrow samples from 143 patients with leukemic phase B-CLPD including 78 cases of CLL, 17 cases of mantle cell lymphoma (MCL), 20 cases of Waldenström macroglobulinemia (WM), 20 cases of follicular lymphoma (FL), 5 cases of marginal zone lymphoma (MZL), 3 cases of B-cell chronic lymphoproliferative disorders-unclassified (B-CLPD-U). We used real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to compare the level of LEF1 mRNA expression in malignant cells from 54 B-CLPD patients, including 44 cases of CLL, 3 cases of MCL, 2 cases of MZL, 3 cases of FL, one case of hairy cell leukemia (HCL), one case of B-cell prolymphocytic leukemia (B-PLL) and 5 cases of healthy donors. All statistical analysis of the data were performed using SPASS version 20.0. Mann-Whitney U test was used to compare the difference between the cohorts. P&lt;0.05 was considered statistically significant. Results: The expression of LEF1 protein was positive in 68 of 78 patients with CLL and 2 of 20 patients with FL, both of whom were pathological low-grade. LEF1 expression of patients with WM, MCL, B-CLPD-U were all negative. The expression level of LEF1 protein in CLL was higher than other B-CLPD patients (including all FL, WM, MCL, B-CLPD-U, P=0.000). Positive expression of LEF1 protein can be used as novel differential diagnostic marker for patients with CLL with higher sensitivity and specificity (sensitivity=87%, specificity=97%).The expression of LEF1 protein was negative in 10 of 78 CLL patients. The 10 patients had atypical immunophenotype. The expression of CD20 was increased, even strong expression in 5 CLL patients. There were 9 CLL with CD200/CD148&lt;1, and 6 cases with CD5 dim expression.The expression level of LEF1 gene in CLL patients was much higher than non-CLL patients and healthy controls (P=0.036, 0.004). The expression level of LEF1 gene had no statistical difference between non-CLL patients and healthy controls (P=0.438). The expression of LEF1 mRNA in 25% CLL patients was lower than healthy controls and 33.3% CLL patients was lower than non-CLL patients. Conclusions: The expression level of LEF1 protein was significantly higher than non-CLL in B-CLPD. We analyzed LEF1 protein may be a novel marker in the differential diagnosis of B-CLPD with higher sensitivity and specificity.The mRNA expression level of LEF1 gene in CLL patients was significant higher than non-CLL patients, but the expression of LEF1 mRNA in 25% CLL patients was lower than healthy controls and 33.3% CLL patients was lower than non-CLL patients. Disclosures No relevant conflicts of interest to declare.

Different Prognostic Significance of a Panel Cytogenetic Aberrations Between Chronic Lymphocytic Leukemia and Other B-Cell Chronic Lymphoproliferative Disorders

Background: B-cell chronic lymphoproliferative disorders (B-CLPD) comprise several entities with similar clinical manifestations, within which chronic lymphocytic leukemia (CLL) account for the majority, others including follicular lymphoma (FL), hairy cell leukemia (HCL), splenic marginal zone lymphoma (SMZL),nodal marginal zone lymphoma (NMZL), gastric mucosa-associated lymphoid tissue (MALT) lymphoma, lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia (LPL/WM), and other unclassified subtypes. Cytogenetic aberrations are now the main prognostic predicators in CLL, with deletion of TP53 or ATM as the adverse predicators, while sole deletion of 13q14 as the favorable one, trisomy 12 and IGH translocation as the neutral factors. The prognostic role of this cytogenetic aberrations panel has not been well established in other B-CLPD excluding CLL.Patients and methods: A panel of DNA probes were used to detect cytogenetic aberrations by fluorescence in situ hybridization (FISH), including RB1/D13S25 at 13q14, ATM at 11q22, TP 53 at 17p13, CEP12 and IGH translocation at 14q32. Totally, 870 patients were enrolled in this study, including 459 CLL patients, 41 SMZL patients, 24 HCL patients, 96 LPL/WM patients, 24 NMZL patients with bone marrows involvement (BMI) ,59 FL patients with BMI and 161 patients without definite subtype classification.Results: The incidence of each cytogenetic aberration in CLL is as follow: 38.0%(169/445) for del 13q14, 11.5% (46/400) for del 11q22, 14.5% (66/455) for del 17p13 , 23.8% (68/286) for +12, and 21.9% (97/443) for t(14q32). While in other B-CLPD excluding CLL is as follow: 10.3% (36/351) for del 13q14, 3.6% (11/305) for del 11q22, 7.9% (30/380) for del 17p13, 12.3% (23/ 187) for +12 and 24.1% (93/386) for t(14q32). The incidence of del 13q14 (p=.000), del 11q22 (p=.000), del17p13 (p=.003) and +12 (p=.002) were higher in CLL than in other B-CLPD. With a median follow-up of 46.0 months (2.0-288.0) in CLL and 39 months (3.0-239.0) in other B-CLPD, the median estimated progress-free survival (PFS) and overall survival (OS) in CLL were 80.0 months (95% CI 67.2-92.7) and 129 months (95% CI 111.5-146.5) respectively, while in other B-CLPD were 84 months (95% CI 71.8-96.1) and 135 months respectively.In CLL, the del 13q (p=.046), del 11q (p=.002) and del 17p (p<.001) were the adverse predictors for the PFS with only del 17p (p<.001) for the OS. There were no significant difference between patients with or without del 13q in aspect of OS (129.0 vs 110.5 months, p=.207). while sole del 13q was the favorable predictor for both PFS (p<.001) and OS (p=.001). Del 11q also indicated poor OS without statistical significance (p=.112). The median PFS for patients with or without +12 was 90 months (95% CI 60.5-119.5) and 93.0 months (95% CI 73.3-112.7) (p=.273), and the median OS was also similar (131.0 months vs. 129.0, p=.921). IGH translocation did not significantly influence both PFS (p=.198) and OS (p=.648).In other B-CLPD except CLL, the del 13q, del 17p and t (14q32) had no impact on both PFS (p=.983, p=.910 and p=.675 respectively) and OS (p=.171, p=.401 and p=.453 respectively). But patients with trisomy 12 had poor PFS (69.0 vs.86.0months, p=.094) and OS (69.0 vs.135.0months, p=.006) compared with those without. Del 11q also adversely impacted the OS (75.0 months vs. not reached, p=.059) but not PFS (62.0 vs. 84.0 months, p=.735).Conclusions: Del 11q and del 17p had the adverse prognostic impact in CLL. However, trisomy 12 was the predictor of poor survival in other B-CLPD which indicated neutral prognostic role in CLL. These results may imply different role of the same cytogenetic aberration in the pathology between CLL and other B-CLPD. DisclosuresNo relevant conflicts of interest to declare.

Gene Expression Profiling Signatures Allow the Identification of Unclassifiable Leukemic B-Cell Lymphoid Neoplasms

Leukemic B-cell chronic lymphoproliferative disorders (B-CLPD) encompass a heterogeneous group of defined disease entities. However, around 10% of the cases cannot be reliably classified based on the current criteria and are considered B-CLPD not otherwise specified (B-CLPD NOS). Few recurrent mutations and genetic alterations have been reported in some entities, but none is specific.We performed gene expression profiling (GEP) to develop a robust GEP-molecular classifier for leukemic B-CLPD. We analyzed purified blood of 189 B-CLPD, including 54 chronic lymphocytic leukemia (CLL), 54 mantle cell lymphoma (MCL), 12 follicular lymphoma (FL), 23 splenic marginal zone lymphoma (SMZL), 4 splenic diffuse red pulp lymphoma (SDRPL), 4 hairy cell leukemia (HCL), 4 HCL-variant (HCL-v), 6 lymphoplasmacytic lymphoma (LPL) and 28 B-CLPD NOS. We used a multiple step approach to build a GEP-array classifier and then analyzed an additional series as a validation cohort by quantitative PCR (qPCR). Mutational analysis of BRAF, MAP2K1, MYD88, NOTCH1, NOTCH2, SF3B1 and TP53 and copy number alterations were studied.In the training set, a supervised analysis clustering revealed that each B-CLPD entity has a specific expression profile. By a multi-step GEP classifier using 43 genes (Table 1) we could classify CLL, SOX11-positive MCL (MCLc), HCL, FL, SOX11-negative MCL (MCLi) and HCL-v cases in six successive stages. However, we were not able to clearly identify distinct signatures for LPL, SMZL and SDRPL. Furthermore, we could classify 36% of B-CLPD NOS cases. Interestingly, the 43-gene signature identified in leukemic samples could also classify the 28 tumor splenic biopsies. Finally, we built a simple 8-gene predictive model using qPCR data (including: FMOD, KSR2, SOX11, MYOF, MME, CCND1, CXCR4, and CAMSAP2) that was used in an independent validation cohort and classified 14% B-CLPD NOS cases. The classification yield increased to 61% and 50%, for GEP-array and qPCR, respectively, when additional morphological, molecular and genetic features were considered.Our findings support the use in a routine base of a simple test as a diagnostic tool that can be applied to help multiparameter interpretation in the classification of leukemic B-CLPD and specially B-CLPD NOS.Table 1Gene signatures (43 genes) and steps used for the GEP-array model.Step modelB-CLPDSpecific signatures1CLLFMOD, KSR2, ADTRP, CLNK, LEF1, FILIP1L, CTLA4, IGSF3, EBF12MCLcSOX11, PLEKHG4B, HDGFRP3, CNN3, PON2, SH3BP4, FCGBP, STMN1, FARP1, DBN1, NREP, NINL, MARCKSL1, MEX3D, CRIM1, KAZN3HCLHPGDS, IL1R2, TJP1, PLOD2, EMP1, NOVA14FLMME, SLC2A5, SMAD1, PRDM155MCLiCCND16HCL-vTUSC1, LRP1B, KCNJ3, NRCAM, MS4A14, FAM129C,CXCR47MiscellaneousLPL-SDRPL-SMZLNo specific genes CLLchronic lymphocytic leukemia; FL: follicular lymphoma, HCL: hairy cell leukemia;HCL-v: hairy cell leukemia variant; LPL, lymphoplasmacytic lymphoma; MCLc: conventionalSOX11-positive mantle cell lymphoma; MCLi: indolent SOX11-negative mantle cell lymphoma;SDRPL, splenic diffuse red pulp lymphoma; SMZL, splenic marginal zone lymphoma.Bold letters indicate some of the genes included in the simple qPCR model. DisclosuresLopez-Guillermo:Roche, Celgene, Mundipharma, Gilead, Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality.

Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56(low) NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56(low) NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94(hi)/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality.

World Health Organization-defined eosinophilic disorders: 2015 update on diagnosis, risk stratification, and management.

The eosinophilias encompass a broad range of non-hematologic (secondary or reactive) and hematologic (primary, clonal) disorders with potential for end-organ damage. Hypereosinophilia has generally been defined as a peripheral blood eosinophil count greater than 1,500/mm(3) and may be associated with tissue damage. After exclusion of secondary causes of eosinophilia, diagnostic evaluation of primary eosinophilias relies on a combination of morphologic review of the blood and marrow, standard cytogenetics, fluorescent in situ-hybridization, flow immunocytometry, and T-cell clonality assessment to detect histopathologic or clonal evidence for an acute or chronic myeloid or lymphoproliferative disorder. Disease prognosis relies on identifying the subtype of eosinophilia. After evaluation of secondary causes of eosinophilia, the 2008 World Health Organization establishes a semi-molecular classification scheme of disease subtypes including 'myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1', chronic eosinophilic leukemia, not otherwise specified, (CEL, NOS), lymphocyte-variant hypereosinophilia, and idiopathic hypereosinophilic syndrome (HES), which is a diagnosis of exclusion. The goal of the therapy is to mitigate eosinophil-mediated organ damage. For patients with milder forms of eosinophilia (e.g. < 1,500/mm(3) ) without symptoms or signs of organ involvement, a watch and wait approach with close-follow-up may be undertaken. Identification of rearranged PDGFRA or PDGFRB is critical because of the exquisite responsiveness of these diseases to imatinib. Corticosteroids are first-line therapy for patients with lymphocyte-variant hypereosinophilia and HES. Hydroxyurea and interferon-alpha have demonstrated efficacy as initial treatment and steroid-refractory cases of HES. In addition to hydroxyurea, second line cytotoxic chemotherapy agents and hematopoietic cell transplant have been used for aggressive forms of HES and CEL with outcomes reported for limited numbers of patients. Although clinical trials have been performed with anti IL-5 (mepolizumab) and anti-CD52 (alemtuzumab) antibodies, their therapeutic role in primary eosinophilic diseases and HES has yet to be established.

Immunoglobulin substitution in patients with indolent non-Hodgkin's lymphoma

Infections are major complications in chronic lymphoproliferative disorders, among them indolent non-Hodgkin's lymphoma (iNHL) including chronic lymphocytic leukemia, follicular lymphoma and multiple myeloma.We report on a retrospective cohort analysis of outpatients with indolent non-Hodgkin's lymphoma who were treated in an oncology / hematology group practice and received intravenous polyvalent immunoglobulin G (IVIG) as supportive care. The aim was to describe the treated iNHL population, the course of therapy and the effects of IVIG administrations on the levels of immunoglobulin G (IgG), the incidence of infections and the survival time. 57 patients with secondary iNHL antibody deficiencies (n = 46) or IgG subclass deficiencies (n = 11) who received IVIG substitution were included. Patients received median 11 IVIG doses with a mean dose of 28 g over a period of median 9.5 months. Mean IgG levels increased with IVIG substitution at about twice and then remained within the normal range. The incidence of infections decreased in 46 % of treated patients. Effects on survival could not be observed. Median overall survival was in the group of substituted patients 124 months (range 7-124), the control group had a median survival time of 96 months (range 3-129) (p = 0.537). IgG levels should be reviewed during IVIG substitution on a regular basis and dosage and intervals should be adjusted individually.

Progressive Transformation of Germinal Centers: Single Center Experience

Background: Progressive transformation of germinal centers (PTGC) is a disease of mostly young adults that generally presents as asymptomatic, localized or generalized lymphadenopathy. Although PTGC usually seen as nodal disease, it can occur at extranodal sites. In the differential diagnosis of chronic lymphadenopathies, infection, lymphoproliferative disorders and progressive transformation of germinal centers should be considered. The most significant entities in the differential diagnosis with PTGC are nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and lymphocyte rich classical Hodgkin lymphoma. PTGC may precede, follow, or be concurrent with lymphomas. Objectives: To review the clinical and pathological features of PTGC, and PTGC’s relationship with lymphoid neoplasia in adult population. Material and Methods: The data of 33 patients who was diagnosed with PTCG at Hospital of Ege University School of Medicine, Pathology Department, between January 2000 and December 2014 were analyzed retrospectively. Clinical and Pathological Features: The median age of the patients was 43.8 years (26-66 years), and of 16 (48.5%) males and 17 (51.5%) females. Biopsy regions were; cervical lymph nodes (LN) 9 (27.3%), axillary LN 9 (27.3%), inguinal LN 6 (18.2%), submandibular LN 3 (9.1%), submental LN 2 (6.1%), parapancreatic LN 1 (3.0%), intraparotid LN 1 (3.0%), supraclavicular LN 1 (3.0%) and intramammarian LN 1 (3.0%). Recurrent PTGC was detected in only one (3.0%) patient. Diffuse large B cell lymphoma (DLBCL) and nodular lymphocyte predominant hodgkin lymphoma (NLPHL) was detected concurrent with PTGC in two cases. Also PTGC was detected 3 years after the diagnosis of DLBCL, NLPHL and T cell rich B cell lymphoma in 3 cases. There was no relapse in these 3 cases and no progression to lymphoma during follow-up of other patients. Conclusion: PTGC is not considered a premalignant condition but development of lymphoma have been reported rarely. If PTGC occurs in the follow of lymphoma cases, follow-ups may be intensified. NLPHL is the most important entity in differantial diagnosis of PTGC and patients should be carefully examined in terms of NLPHL.

Analysis and clinical correlation of infiltrating cytotoxic T cells in Hodgkin Lymphoma tumor samples using flow cytometry

Background: Progressive transformation of germinal centers (PTGC) is a disease of mostly young adults that generally presents as asymptomatic, localized or generalized lymphadenopathy. Although PTGC usually seen as nodal disease, it can occur at extranodal sites. In the differential diagnosis of chronic lymphadenopathies, infection, lymphoproliferative disorders and progressive transformation of germinal centers should be considered. The most significant entities in the differential diagnosis with PTGC are nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and lymphocyte rich classical Hodgkin lymphoma. PTGC may precede, follow, or be concurrent with lymphomas. Objectives: To review the clinical and pathological features of PTGC, and PTGC’s relationship with lymphoid neoplasia in adult population. Material and Methods: The data of 33 patients who was diagnosed with PTCG at Hospital of Ege University School of Medicine, Pathology Department, between January 2000 and December 2014 were analyzed retrospectively. Clinical and Pathological Features: The median age of the patients was 43.8 years (26-66 years), and of 16 (48.5%) males and 17 (51.5%) females. Biopsy regions were; cervical lymph nodes (LN) 9 (27.3%), axillary LN 9 (27.3%), inguinal LN 6 (18.2%), submandibular LN 3 (9.1%), submental LN 2 (6.1%), parapancreatic LN 1 (3.0%), intraparotid LN 1 (3.0%), supraclavicular LN 1 (3.0%) and intramammarian LN 1 (3.0%). Recurrent PTGC was detected in only one (3.0%) patient. Diffuse large B cell lymphoma (DLBCL) and nodular lymphocyte predominant hodgkin lymphoma (NLPHL) was detected concurrent with PTGC in two cases. Also PTGC was detected 3 years after the diagnosis of DLBCL, NLPHL and T cell rich B cell lymphoma in 3 cases. There was no relapse in these 3 cases and no progression to lymphoma during follow-up of other patients. Conclusion: PTGC is not considered a premalignant condition but development of lymphoma have been reported rarely. If PTGC occurs in the follow of lymphoma cases, follow-ups may be intensified. NLPHL is the most important entity in differantial diagnosis of PTGC and patients should be carefully examined in terms of NLPHL.