Absolute Lymphocytosis Research Articles

Polyclonal B-cell lymphocytosis with binucleated lymphocytes (PPBL).

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare and recently described entity. The review of the literature show PPBL is diagnosed predominantly but not exclusively in women, usually smokers. PPBL is recognized by a moderate, chronic and absolute lymphocytosis (>4 × 10(9)/l) in the peripheral blood. In 10% of cases without lymphocytosis, the PPBL diagnosis has to be suggested by peripheral blood examination showing in all cases atypical binucleated lymphocytes. A polyclonal serum IgM is also associated and HLA-DR7 expression is present in most cases. Contrary to B-cell chronic lymphoproliferative disorders (B-CLPD), peripheral B cells are polyclonal with kappa and lambda light-chain expression and no clonal rearrangement of immunoglobulin heavy chain genes is usually demonstrated. The detection of an extra isochromosome for the long arm of chromosome 3 +i(3)(q10) has to be considered as a specific marker of PPBL. We performed conventional cytogenetic analysis (CCA) in 111 patients with typical PPBL we followed-up more than 4 years. +i(3q) was detected in 34% (33/98), PCC in 8% (8/98) and both abnormalities in 31% (30/98). CCA showed neither +i(3q) nor PCC in 28% (27/98). Fluorescence in situ hybridization (FISH) was also performed in 84 cases and +i(3q) was detected in 71% (60/84). When combining both procedures in 84 patients, +i(3q) was detected in 17 patients with negative CCA and was confirmed in 43 patients with positive CCA. CCA and FISH were both negative in 24 cases. Whether patients with PPBL are at increased risk of hematological malignancy remains unclear. After a median follow-up of 4.4 years, most PPBL patients presented a stable clinical and biological course. Six patients died from pulmonary cancer, myocardial infarction, cerebral aneurysm rupture or diffuse large B-cell lymphoma. Two patients had IgM monoclonal gammopathy of undetermined significance (MGUS) at the time of PPBL diagnosis and two other patients developed IgM MGUS respectively 12 and 22 years after PPBL diagnosis. A malignant non Hodgkin's lymphoma (NHL) appeared in 3 additional patients: two patients presented diffuse large B cell lymphoma and 1 patient a splenic marginal zone lymphoma. In conclusion, the possibility of PPBL to evolve toward a clonal proliferation, malignant lymphoma or secondary solid cancer lead us to consider PPBL not as a benign pathology. We recommend a careful and continued clinical and biological long-term follow-up in all PPBL patients.

MYC translocation in chronic lymphocytic leukaemia is associated with increased prolymphocytes and a poor prognosis.

Chromosomal translocations that involve MYC, characteristic of Burkitt lymphoma, are rare in chronic lymphocytic leukaemia (CLL). We report the clinical, morphological, immunophenotypic, cytogenetic and molecular genetic features of eight CLL cases with MYC rearrangement. The patients, five men and three women (median age, 71 years) had bone marrow involvement and an absolute peripheral blood lymphocytosis; five had lymphadenopathy; seven had splenomegaly. Prolymphocytes were increased (>/=10%) in all cases. Six cases were classified as CLL with increased prolymphocytes (CLL/PL; prolymphocytes 10-55%), and two were classified as CLL in prolymphocytic transformation (CLL/PT; prolymphocytes >55%). All cases co-expressed CD5, CD19, and CD23; five of eight expressed ZAP-70. Of seven cases tested, four had mutated and three had unmutated IGHV genes. Conventional cytogenetic studies demonstrated t(8;14)(q24.1;q32) in five cases, t(8;22)(q24.1;q11) in two cases, and t(2;8)(p12;q24.1) in one case. Seven cases contained additional chromosomal abnormalities. All patients received combination chemotherapy. Two developed Epstein-Barr virus (EBV)-associated diffuse large B-cell lymphomas (DLBCL) that were clonally unrelated to the CLL. At follow-up, two patients are alive, four died of underlying disease, one died of EBV-associated DLBCL, and one died of an unrelated cancer. In summary, MYC rearrangement, which occurs rarely in CLL patients, is associated with increased prolymphocytes, complex cytogenetic abnormalities, and a poor prognosis.

Relationship of Lymphocyte-Neutrophil Ratio to Heterophile Antibody Result

Abstract The heterophile antibody test (HAT) is commonly ordered at the same time as a complete blood cell (CBC) count in cases of suspected infectious mononucleosis. An absolute lymphocytosis with the presence of greater than 10% atypical lymphocytes is a good predictor of a positive HAT. We retrospectively studied patients who underwent HAT and had a simultaneous CBC count during an 8.5-year period. The ratio of the absolute number of lymphocytes to the absolute number of neutrophils (L/N) was determined to see if this measure could be useful in distinguishing those most likely of having a negative HAT. A total of 5104 patient samples were analyzed of which 501 (9.8%) were HAT positive. The L/N ratio was less than 0.5 in 2214 patients (43.4%), and in this group, only 46 (2.1%) were HAT positive. In the group whose L/N ratio was greater than or equal to 0.5, the HAT positivity rate rose to 15.7% (455/2888), for a relative increase of 60%. In the HAT-positive group, 153 (30%) did not demonstrate an absolute lymphocytosis. Restricting HAT to those having a CBC count with an L/N ratio greater than or equal to 0.5 will increase the HAT positivity rate and lead to a reduction of HAT performed.

Using peripheral smear review, age and absolute lymphocyte count as predictors of abnormal peripheral blood lymphocytoses diagnosed by flow cytometry

Absolute lymphocytosis in the elderly raises the possibility of malignancy and generally warrants further investigation. To better correlate clinical variables with the frequency of neoplastic lymphoid processes in this population, we retrospectively reviewed archived flow cytometric analyses from peripheral blood specimens on patients of 50 years of age and older that had been deemed suspicious for a lymphoproliferative process after peripheral smear review. Age, absolute lymphocyte count (ALC), white blood cell count and relative lymphocyte count were correlated with the results of flow cytometry. Of 71 total cases, 42 (59%) had an abnormal immunophenotype. Independent variables that showed significant differences between normal and abnormal immunophenotype were mean age (p = 0.001) and ALC (p = 0.0032). We combined age and absolute lymphocyte count variables to look for the best possible cutoff values to predict the likelihood of an abnormal immunophenotype. ALC cutoff values of ≥4 × 109 cells/L for patients over 67 years of age, and >6.7 × 109 cells/L for patients between 50 and 67 years of age, had a high sensitivity for detecting an abnormal immunophenotype.

Concomitant Diagnosis of Chronic Myeloid Leukemia and B-Cell Chronic Lymphocytic Leukemia in a 57-Year-Old Female Patient, with Good Therapeutic Response Exclusively to Imatinib in Both Myeloid and Lymphoid Clones.

We describe the clinical, cytogenetic, hystopathologic, immunophenotypic and molecular findings of a rare case of Philadelphia positive chronic myeloid leukemia (Ph+ CML) and B-cell chronic lymphocytic leukemia (B-CLL) occurring simultaneously at diagnosis, in a 57-year-old female patient. In the present case, the patient showed at diagnosis leukocytosis (48.000/ul) with myelocytes and metamyelocytes along with well-differentiated neutrophils, but also with well differentiated lymphocytosis (28.992/ul) in peripheral blood (PB) and also in bone marrow (BM) aspirate cytology. FACS cytometry exhibited clearly two predominant separated cellular populations and the immunophenotype profile of the lymphoid component in BM and PB was CD5+, CD20+, CD23+, with light chain clonal restriction. The BM hystopathological analysis showed two distinct cellular populations, one lymphoid well differentiated CD5+/CD20+, and other myeloid, in several stages of differentiation. Cytogenetic analysis with unstimulated bone marrow culture confirmed the presence of Philadelphia chromosome but with additional translocation involving chromosomes 9, 10 and 22. Molecular studies showed the evidence of BCR-ABL positivity and the B-cell clone was documented by the presence of a clonal heavy chain immunoglobulin rearrangement. The patient was treated with imatinib mesylate 400mg/day, achieving complete cytogenetic response at the fourth month, and complete molecular response (nested PCR) of the BCR-ABL component at the sixth month of treatment. Besides, the absolute lymphocytosis was gradually reduced during the treatment exclusively with imatinib, achieving normal absolute values at the sixth month. This seldom described case of simultaneous occurrence of these two rare chronic hematological malignancies, brings interesting questions about the possible cellular origin and the correspondent mechanisms of clonal expansion. The therapeutic response of the B-cell lymphoid clone exhibited, brings also a possible relationship with the observation that the c-Abl could have enhanced expression in B-CLL cells, as described by Ke Lin et al, in which the c-Abl kinase activity could be inhibited by imatinib mesylate.

Expression Levels of a Single Gene, Lymphoid Enhancer Binding Factor 1, Discriminates CLL B-Cells from Other B-Cell Malignancies.

B-cell chronic lymphocytic leukemia (CLL), the most common adult leukemia, is a clonal disease manifested by an absolute lymphocytosis. Previously we identified genes capable of sorting and predicting the B-cells of patients with CLL or monoclonal B-cell Lymphocytosis (MBL) from aged matched controls. Using gene expression profiling, we and others have identified genes transcribed at significantly different levels between CLL patients and normal subjects. From these genes RT-QPCR verification was performed, in triplicate or more, on 22 gene candidates. A gene panel of seven genes, significantly different between CLL and normal individuals was chosen: FMOD, CKAP4, PI3Kc2b, LEF1, PFTK1, Bcl2 and GPM6a (in order of Receiver Operating Characteristic [ROC] concordance). This RT-QPCR verification step was based upon the comparison of freshly isolated PBMCs from CLL patients and age matched normal subjects after enriching for B cells with negative selection. The panel was then used to blindly categorize and predict the relationship of RNA from apparently normal subjects, some with and some without MBL, to the mRNA of patients with CLL as well as normal subjects, presumably without MBL. Since the numbers of cells within the expanded clones from individuals with MBL, after positive selection was limiting, RNA was amplified utilizing an RNA whole genome amplification approach and compared to similarly amplified RNA after negative selection; CLL cells, B-cells from normal age matched controls, and cord blood-derived B1 cells, enriched further by CD5 expression with positive selection. Using the Bayesian relevance determination method, a novel computer-learning tool to provide a probabilistic model, 56 samples were identified correctly at the rate of 100%. FMOD and PI3Kc2b were determined to be optimal at predicting blinded samples. In order to make the panel more clinically feasible, a universal reference RNA standard was used as a control, eliminating the need for pairing samples with age-matched controls. Analysis of this new model led to the discovery that a single gene; lymphoid enhancer binding factor 1 (LEF1), was not only capable of sorting CLL B cells from age-matched controls at a 100% rate (n=40) but it was also capable of predicting CLL B cells from other B cell malignancies (n=15). Verification of the protein expression levels for these genes is ongoing. The RNA expression levels of the genes in this panel provides a novel way to identify cell populations and gene expression patterns that change during the transition from B-cell clonal expansions that occur physiologically from those that occur among pre-leukemic and leukemic B-cell populations. The addition of a standard reference RNA enhances the clinical application of this gene panel. These data strongly suggest a unique role for LEF1 in CLL.

HTLV-1 Adult T Cell Leukemia-Lymphoma Presenting with Refractory Hypercalcemia, Cranial Neuropathy and Diagnosis by Flow Cytometry and Cytogenetic Findings.

Introduction: ATLL) is a postthymic lymphoproliferative neoplasm of T cells caused by human T-cell lymphotropic virus (HTLV-1). Most cases are found in Japan, the Caribbean basin, and West Africa.ATLL is essentially a disease of adults, characterized by generalized lymphadenopathy, hepatosplenomegaly, skin lesions, and hypercalcemia. The prognosis of most patients is quite poor, with a median survival time of only 13 months, even with combination chemotherapy. The acute form had a significantly shorter survival (2 months) than lymphoma type (13 months)Advanced performance status, high lactic dehydrogenase, age > 40 years, increased total involved lesions and hypercalcemia, are associated with shortened survival (p<0.01). Hypercalcemia is observed in >80% of ATL. CASE Report:A 43 years old carribean male presented with refractory hypercalcemia 18 g/dl wihtout hematological manifestations,lymphadenopathy or hepatosplenomegaly. He had normal skeletal survey,serum immunoglobulins,immunofixation.The patient also had normal PTH,PTHrp,Vitamin D3,angiotensin converting enzyme levels and 24 hour urinary calcium.The patient had Hemoglobin 13.5 and absolute lymphocytosis 5000–6000/cubic mm.Peripheral smear showed clover T cells Bone marrow had normal morphology but flow cytometry revealed T cells immunoreactive for CD3, CD4,CD8, CD5, and CD25 and were nonimmunoreactive for CD7 .The patient developed visual disturbances, swllowing difficulty and facial palsy during hospital stay.MRI brain was normal but CSF flow cytometry was positive for CD2, CD3, CD4, CD8,CD5, and CD25 and were negative for CD7. Cytogenetic studies showed complex karyotype with: 47, XY,add(4)(p12),+add(4)(p12),add(5)(q11.2),add(7)(q31),–12,Add(12)(q22),der(13)t(5;13)(q11.2;q22),add(14)(q32),add(17)(q23) +mar1(11)/48,idem,+mar1(5)/46,XY(4).A Hyperdiploid male karyotype with monosomy for chromosome No.12,unbalanced translocation involving the long chromosome Nos. 5 and 13, additional material on long arm of 5,7, 12, 14(14q+chromosome) and 17 Complex karyotypes with numerical and structural abnormalities with six or more breaks involving the long arms of chromosomes Nos. 1, 3,6,7,12 and 14q 32 and the short arm of chromosome Nos. 10, 17 and 21 are reported in ATLL .T cell gene rearrangement confirmed the diagnosis.The patient had history of Strongyloides stercoralis infection 8 years ago.He was HIV negativebut had HTLV-1 antibodies(gp19,gp21).He was treated with EPOCH regimen along with intrathecal treatment with normal repeat cytology and CSF flow cytometry.The patientis planned for allogeneic transplant after completion of chemotherapy.Discussion: Adult T-cell lymphoma virus type 1, is implicated diseases including HTLV 1 associated myelopathy,Strongyloides stercoralis hyperinfection, and adult T leukemia/lymphoma. The virus activates a subset of T helper Th1cells with suppression of the Th2 lymphocytes and a reduction of Th2 cytokine production of IL-4, IL-5, IL-10 and IL-13 with an inadequate immune response to invading parasitic infections such as Strongyloides stercoralis. Dual positivity for CD4 and CD8 cells thus confers better prognosis. [Display omitted]

Diagnostic, pronostic et traitement chez les patients avec une leucémie lymphoïde chronique

B-Chronic lymphocytic leukaemia (CLL) is the most frequent of chronic leukaemias. It is defined by a durable absolute lymphocytosis (> 4 × 10 9/l) with small mature lymphoid cells in the peripheral blood. Immunological score established on five criterias (positivity of CD5 and CD23, negativity of the FMC7 and weak expression of sIg and CD22 or CD79b) is four or five. The Binet classification defines three stages of different evolution: the stage A (65% of the cases) and B (28%) without or with three areas involved at least, in the absence of anaemia or thrombopenia and the stage C with anaemia (haemoglobin lower than 10 g/dl) or thrombopenia (platelets lower than 100 × 10 9/l). Patients with stage A will a few progress quickly and conversely others will remain stable with no evolution for very long periods. Four markers with prognosis value were recently identified and allow to adopt new therapeutic strategies according to the presence or not of these markers. These factors are lymphocytes doubling time, serum thymidine kinase, cytogenetic abnormalities including del(11) and del(17), +12 and del(13), and the immunoglobulin mutational profile. The therapeutic strategies evolved these last years and call, if criteria of gravity are present, to chemotherapy (purines analogs associated or not with cyclophosphamid) and immunotherapy (anti-CD20 and/or anti-CD52 monoclonal antibody).

Spleenic lymphoma villous lymphocytes: Case report and literature review

Splenic lymphoma with villous lymphocytes (SLVL) is a rare but recognized distinct disease entity among chronic B lymphoproliferative disorders. It is frequently misdiagnosed as chronic lymphocytic leukaemia, (CLL) Prolymphocytic leukaemia or Hairy Cell leukaemia. Few cases have been reported worldwide. The case records of a sixty year old Nigerian male with a splenic lymphoma and a review of the literature on the subject using MEDLINE, other internet sources and manual library search was utilized. RESULT A sixty-year-old male with splenic lymphoma with villous lymphocytes, seen at the Ahmadu Bello University Teaching Hospital Zaria is presented. However review of the blood film, bone marrow and Splenic aspirates showed absolute lymphocytosis, consisting of "villous" lymphocytes. He was commenced on an alkylating agent and a glucocorticoid, with partial remission in the first three months but was lost to follow up. Adequate morphologic evaluation is advocated particularly in the resource limited settings were Cytogenetics, immunohistochemistry and immunophenotyping are not available.

Cytogenetic Aberrations and ImmunoglobulinVHGene Mutations in Clinically Benign CD5– Monoclonal B-Cell Lymphocytosis

The finding of monoclonal B-cell lymphocytosis (MBL) raises questions on the nature of clonal cell expansion and its risk of progression. We identified and characterized 7 cases of clinically benign clonal B-cell lymphocytosis. The clonal lymphocytes were clearly of CD5- and non-chronic lymphocytic leukemia (CLL) phenotype. All cases had mild to moderate absolute lymphocytosis. The clonal population accounted for 95% to 99% of B cells. For a follow-up period of 4 to 16 years, clonal lymphocytosis was persistent but virtually not progressing. Patients' conditions remained clinically stable and asymptomatic. The clonal populations had somatic hypermutations of the VH gene in 6 cases, indicating a germinal center or post-germinal center B-lymphocyte origin. Clonal cytogenetic aberrations were found in 5 of 6 cases, with 2 clones bearing isochromosome 17q that resulted in loss of p53 and 2 other clones with 7q abnormalities. By the presence of absolute lymphocytosis, this series differs from MBL cases identified by sensitive flow cytometry in normal populations. The phenotypic profiles are distinct from that of benign CLL. We suggest these CD5-B-cell lymphocytosis cases may represent an intermediate condition between covert clonal expansions and overt malignancy.

Outline for Writing an Article for Current Treatment Options in Oncology: Splenic Lymphoma with Villous Lymphocytes

Two subtypes of splenic marginal zone lymphoma (SMZL) are identified in the World Health Organization (WHO) classification: SMZL without villous lymphocytes and SMZL with villous lymphocytes in the peripheral blood (SLVL). SLVL is a rare leukemic and indolent B-cell chronic lymphoproliferative disorder (B-CLPD) that we have to differentiate from hairy cell leukemia (HCL), B prolymphocytic leukemia (B-PLL) and follicular lymphoma (FL). Morphological examination associated with immunophenotyping is, in most cases, likely to distinguish these CD5 negative entities. However, the diagnosis can be difficult to make on morphological criteria, especially in patients without absolute lymphocytosis. Based on histologic, cytogenetic and molecular studies, SLVL emerges as a distinct entity. SLVL has a relatively clinical benign course but a few patients could require treatment, because of a symptomatic splenomegaly and/or a severe cytopenia. In symptomatic patients HCV negative, the frontline treatment remains questionable. Splenectomy, regarded as the most effective treatment, could be required for diagnostic purposes: however, relapse occur in 30% of cases. Fludarabine (FDR), a purine analogue and deoxycoformycin (DCF) can induce a maintained response in a substantial proportion of patients with SLVL and could be used as a first line treatment. In HCV + SLVL patients, antiviral treatment using alpha interferon and ribavirin can induce regression of SLVL.

Asymptomatische 32-jährige Raucherin mit persistierender Lymphozytose

A 32 year old female smoker (20 pack years) presented with an asymptomatic lymphocytosis of 13,000/nl and splenomegaly. The patient's blood smear showed an absolute lymphocytosis with 65% atypical lymphocytes. A total of 1% of the lymphocytes were bilobulated. Bone marrow histology and immunphenotyping of blood and bone marrow excluded leukemia and non-Hodgkin's lymphoma. IgH-CDR-3 PCR analysis revealed a polyclonal pattern. In summary, a persistent polyclonal B-cell-lymphocytosis (PPBL) was diagnosed. The exact etiology of PPBL is still unclear, however, it is associated with a polyclonal raise in the lymphocyte count of CD27+IgD+-memory-B-lymphocytes due to a defect in apoptosis signaling and leukocyte homing to secondary lymphoid tissues. An association with cigarette smoking is obvious since all patients are smokers. From all published cases, only two developed a malignancy with an uncertain association with PPBL. We have been monitoring our patient for 6.5 years without any evidence of the development of a lymphoma.

Unusual expression of CD94 on CD8+ TCR-αβ T cells in infectious mononucleosis

Infectious mononucleosis, caused by primary Epstein-Barr virus (EBV) infection, is usually a benign, self-limited lymphoproliferative disorder. We report a case of a 21-year-old woman who presented with fever, sore throat, severe neutropenia, and absolute lymphocytosis with atypical lymphocytes. In situ hybridization for EBV-encoded small RNA performed on the marrow aspirate clot specimen demonstrated scattered positive cells. EBV serology was compatible with primary infection. Flow cytometry immunophenotypic studies performed on aspirate material revealed a profoundly expanded population of CD8+ T-cell receptor (TCR)-alphabeta T cells with uniform expression of CD94. No evidence of a monoclonal T-cell population was found as assessed by V(beta) use with flow cytometry and by TCR gamma-chain gene rearrangement using a polymerase chain reaction method. Uniform expression of CD94 in an exuberant reactive proliferation of CD8+ TCR-alphabeta T cells in infectious mononucleosis has not been reported previously, and combined with atypical morphology might be misinterpreted as a malignant neoplasm.

Which observations from the complete blood cell count predict mortality for hospitalized patients?

Information on the prognostic utility of the admission complete blood count (CBC) and differential count is lacking. To identify independent predictors of mortality from the varied number and morphology of cells in the complete blood count defined as a hemogram, automated five cell differential count and manual differential count. Retrospective cohort study and chart review. Wishard Memorial Hospital, a large urban primary care hospital. A total of 46,522 adult inpatients admitted over 10 years to Wishard Memorial Hospital-from January 1993 through December 2002. None. Thirty-day mortality measured from day of admission as determined by electronic medical records and Indiana State death records. Controlling for age and sex, the multivariable regression model identified 3 strong independent predictors of 30-day mortality-nucleated red blood cells (NRBCs), burr cells, and absolute lymphocytosis-each of which was associated with a 3-fold increase in the risk of death within 30 days. The presence of nucleated RBCs was associated with a 30-day mortality rate of 25.5% across a range of diagnoses, excluding patients with sickle-cell disease and obstetric patients, for whom NRBCs were not associated with increased mortality. Having burr cells was associated with a mortality rate of 27.3% and was found most commonly in patients with renal or liver failure. Absolute lymphocytosis predicted poor outcome in patients with trauma and CNS injury. Among patients admitted to Wishard Memorial Hospital, the presence of nucleated RBCs, burr cells, or absolute lymphocytosis at admission was each independently associated with a 3-fold increase in risk of death within 30 days of admission.

The t(14;19)(q32;q13)‐positive small B‐cell leukaemia: a clinicopathologic and cytogenetic study of seven cases

The t(14;19)(q32;q13), involving the BCL3 locus at chromosome 19q13 and the immunoglobulin heavy chain gene at 14q32, is a rare recurrent cytogenetic abnormality identified in B-cell neoplasms, most of which have been classified as chronic lymphocytic leukaemia (CLL) in the literature. We describe the clinicopathological, immunophenotypic and cytogenetic findings in seven patients with B-cell neoplasms associated with t(14;19)(q32;q13). There were five men and two women, with a median age of 48 years (range 33-68). All had absolute lymphocytosis, six had lymphadenopathy, and one had splenomegaly. Lymphocytes in blood and bone marrow aspirate smears were predominantly small and cytologically atypical. Flow cytometric immunophenotyping showed an atypical immunophenotype with low CLL scores. The growth pattern in bone marrow biopsy specimens was interstitial to diffuse; immunohistochemical stains were positive for bcl3 and negative for cyclin D1. Lymph node biopsy specimens of two patients revealed total architectural effacement by neoplasm with proliferation centres. In addition to t(14;19), cytogenetic studies demonstrated trisomy 12 in five patients. These results suggest that B-cell neoplasms with the t(14;19)(q32;q13) present frequently as leukaemia composed of small B-lymphocytes and share many features with CLL. However, these neoplasms also differ from CLL cytologically and in their immunophenotype.

Concurrent Treatment with Denileukin Diftitox (DD) To Deplete T-Regulatory Cells Enhances Rebound Lymphocytosis and Eosinophilia in Patients Treated with High-Dose IL-2 (HDIL-2) for Metastatic Renal Cell Cancer (MRCC).

Background: CD4+CD25+hi T cells (Treg) play a suppressive role in immune regulation. DD is an IL-2 receptor specific cytotoxin. We postulated depletion of Treg with DD may enhance immune effector cell populations after HDIL-2 treatment, including rebound lymphocytosis and also eosinophilia which has been reported to be involved in immune response to neoplasm (Mattes J Exp Med 197: 387, 2003).Methods: In this pilot study, 12 pts (8 male, median age 58 yrs) with MRCC were tx with HDIL-2 and DD in different schedules to determine safety and effect on immune response as manifested by changes in Treg, peak lymphocyte, and eosinophil counts. Pts were treated with IL-2 600,000 IU/kg Q8H on days (d) 1–5 and 15–19. Three (group A) and 4 (group B) pts were given 6 and 9ug/kg daily on d8–10 respectively, while 5 (group C) pts received 9ug/kg of DD on d −4 to −2. Nine (group D) pts with metastatic melanoma who received HDIL-2 as above but without DD were included as controls. Flow cytometry was done on days −4, 1,8,10,15,22 for group C and on days 1,8,10,15,22 for groups B, and D. CBC was obtained concurrent or within 24 hours of flow cytometry. Group A pts were evaluated for safety only and were excluded from analysis.Results: Prior to enrollment, all pts had undergone nephrectomy and four patients received interferon-alpha. One pt from group B withdrew from study and was not included in analysis. Administration of DD resulted in a median decline of 25% in Treg number (not significant). DD given before HDIL-2 was associated with a greater increase in Treg post HDIL-2. In Group C there was an increase of rebound median Treg count of 0.88k/ul compared with 0.060k/ul in group B (p=0.025). Absolute lymphocytosis was higher in the combined group getting DD compared to control (median maximal increase of 7.6 vs 4.7 k/ul, respectively) although the difference did not reach statistical significance. However, group C pts had a greater increase in absolute lymphocytosis than did group B pts in which absolute lymphocytosis actually decreased (median increase 10.6 vs. median decrease 0.4 k/ul, p=0.025). A higher peak level of eosinophilia was noted in groups B and C compared with group D (mean increase of 10.5 vs. 4.0 k/ul p=0.2). Group C had a greater peak eosinophilia than group B (11.2 vs 2.2 k/ul p=0.053) Toxicity was manageable and consistent with those seen with HDIL-2. Median HDIL-2 dose given was 21 (range, 14–28). No clinical responses were observed. Of 11 pts included in the analysis 1 pt from group A expired 68 weeks after enrollment. All remaining patients are alive. Survival from enrollment ranges from 11 to 93 weeks.Conclusion: Overall, the combination of DD and HDIL-2 results in a stimulatory effect as manifested by increased rebound lymphocytosis and eosinophilia compared to HDIL-2 alone. Administration of DD in conjunction with HDIL-2 was associated with a rebound in Treg that may be schedule and dose dependent. The results suggest an enhanced immune stimulatory effect as manifested by lymphocytosis and peak eosinophilia in group C. However, this stimulatory effect also extends to Treg that may prove detrimental clinically. Further exploration of these effects in immunotherapy naïve patients would be beneficial.

Cytogenetic Aberrations and Ig VH Gene Mutations of Clinically Benign CD5-Monoclonal B-Cell Lymphocytosis (MLUS).

Clonal expansion of lymphocytes is a hallmark of lymphoproliferative disorders. However, the finding of clonal lymphocytes may not be diagnostic of a defined lymphoid malignancy. In this study, we had an opportunity to identify and characterize a series of 7 patients with clinically benign clonal B-cell lymphocytosis. The clonal lymphocytes were clearly of CD5− and non-CLL phenotype. All patients were elderly and identified with a mild to moderate absolute lymphocytosis (lymphocytes range 3.6–9.4 ×109/L). The clonal population accounted for 95–99% of total B cells. For a follow-up period of 4–16 years, clonal lymphocytosis was persistent but virtually not progressing. All patients remained clinically stable and totally asymptomatic. The clonal CD5− B cells were shown to have somatic hypermutations of VH gene in 6 of the 7 patients, indicating a cell origin of germinal center B lymphocytes. Karyotypic aberrations were found in 5 of 6 patients examined. Two clones were found to have an isochromosome 17q, one as the sole abnormality and one as part of a complex karyotype. The loss of the short arm of chromosome 17 resulted in loss of p53, which was confirmed by the FISH analysis in both cases. The finding of p53 loss is unusual in such a benign condition. Two other clones were shown to have 7q abnormalities; one 7q31–34 deletion and one t(2;7)(p11;q22), the latter is impliated in dysregulation of the CDK6 gene. Even though deletion of 7q is strongly associated with, and t(2;7) is confined to splenic marginal zone lymphoma (SMZL), neither patients were hematologically or clinically diagnostic for SMZL. It remains to be determined whether additional cytogenetic and molecular changes may occur to lead to progression to a clinical malignancy. The patients in this study differ from those clonal CD5− monoclonal B-cell populations identified by sensitive flow cytometry in the normal population, by the presence of absolute lymphocytosis. In addition, the phenotypic profile is distinct from that of benign variant CLL. These clinically benign lymphocytoses may represent an intermediate between covert clonal expansion and overt malignancy.

A new automated flow cytometry data analysis approach for the diagnostic screening of neoplastic B-cell disorders in peripheral blood samples with absolute lymphocytosis

Currently, multiparameter flow cytometry immunophenotyping is the selected method for the differential diagnostic screening between reactive lymphocytosis and neoplastic B-cell chronic lymphoproliferative disorders (B-CLPD). Despite this, current multiparameter flow cytometry data analysis approaches still remain subjective due to the need of experienced personnel for both data analysis and interpretation of the results. In this study, we describe and validate a new automated method based on vector quantization algorithms to analyze multiparameter flow cytometry immunophenotyping data in a series of 307 peripheral blood (PB) samples. Our results show that the automated method of analysis proposed compares well with currently used manual approach and significantly improves semiautomated approaches and, that by using it, a highly efficient discrimination with 100% specificity and 100% sensitivity can be made between normal/reactive PB samples and cases with B-CLPD based on the total B-cell number and/or the sIgkappa+/sIglambda+ B-cell ratio. In addition, the method proved to be able to detect the presence of pathologic neoplastic B-cells even when these are present at low frequencies (<5% of all lymphocytes in the sample) and in poor-quality samples enriched in 'noise' events.

Serum levels of syndecan-1 in B-cell chronic lymphocytic leukemia: Correlation with the extent of angiogenesis and disease-progression risk in early disease

Syndecan-1 is a transmembrane proteoglycan generally not expressed in mature B-cell neoplasias like chronic lymphocytic leukemia (CLL). Moreover, information dealing with the evaluation of soluble syndecan-1 in CLL are lacking. We measured syndecan-1 concentrations in serum drawn at the time of diagnosis from 67 B-cell CLL patients (Binet stage A, 46; stage B, 7; stage C, 14). For this purpose a syndecan-1 enzyme-linked immunosorbent assay (ELISA, Diaclone, Besancon, France) was used. Detectable levels of syndecan-1 were found in all patients, although serum concentrations were significantly lower in CLL patients in comparison to age- and sex-matched controls (P = 0.02; Mann-Whitney test). No correlation was found with Binet clinical stages (P = 0.796), β2-microglobulin (P = 0.923), hemoglobin level (P = 0.605), platelet count (P = 0.992) and lymphocyte doubling time (P = 0.709). Only an association with absolute peripheral blood lymphocytosis (PBL) (P = 0.01) and LDH (P = 0.05) could be detected. Serum levels of syndecan-1 did not parallel those of several angiogenic cytokines such as vascular endothelial growth factor (VEGF) (P = 0.963), basic fibroblastic growth factor (FGF-2) (P = 0.216), angiogenin (P = 0.478), metalloproteinase-9 (MMP-9) (P = 0.125) as well as bone marrow (BM) microvessel density (P = 0.110). The same applied with adhesion molecules such as CD54 (P = 0.233), CD108 (P = 0.799), CD44 (P = 0.816) and CD31 (P = 0.508). Interestingly, the inverse correlation (r = −0.4967; P = 0.03) between serum concentrations of syndecan-1 and plasma levels of stromal derived growth factor-1 (SDF-1) is in keeping with the different function, respectively, pro- and anti-apoptotic, of these molecules. In 46 Binet stage A patients, serum levels of syndecan-1 were further evaluated as a dichotomous variable with respect to progression-free survival (PFS), an end-point surrogate for overall survival in early B-cell CLL. The best separation of curves was seen with a cut-off point at the median value of syndecan-1 (i.e. 36.5 pg/ml). Median PFS was not reached in the patient group with low syndecan-1, compared to a median of 34 months observed in the remaining patients (P = 0.018; HR = 0.208; 95% CI = 0.115 – 0.816). At the multivariate analysis performed including variables significant in the univariate analysis [i.e. PBL (P = 0.03) and syndecan-1 (P = 0.01)], only syndecan-1 retained a trend of significance (P = 0.08).Despite the pro-angiogenic activity of syndecan-1 which mediates FGF-2 binding and activity, no correlation with either angiogenic cytokines or the extent of BM angiogenesis was found in CLL. The inverse correlation with plasma levels of SDF-1 suggests an involvement in the processes leading to apoptosis. Finally, our results highlight the involvement of syndecan-1 in the mechanisms of disease-progression of early CLL.

High Incidence of Ancestral HLA Haplotype 8.1 and Incomplete DH-JH Immunoglobulin Rearrangement in Persistent Polyclonal B-Cell Lymphocytosis.

Four women aged from 48 to 51 years with persistent polyclonal B-cell lymphocytosis (PPBCL) were studied for the clonality of B-cell population and HLA haplotype. All patients were smokers, one presented an immune deficiency syndrome due to decreased IgG and IgA levels, the others were asymptomatic. All patients presented serum increase of polyclonal IgM (5.55 – 8.12 g/L), absolute peripheral lymphocytosis ranging from 6100 to 10500/μL with elevated proportion of B cells, the percentage of CD5/CD19 lymphocytes < 5%, and binucleated/bilobulated lymphocytes accounting for 5–9% of nucleated cells on the blood smear. In one patient, cytogenetic study revealed trisomy 3 in 4 out of 40 metaphases, +18 in 2 cells, and i(18q), +15, t(13,14) in single cells. In the remaining patients no clonal chromosomal abnormalities were found. All patients were HLA typed by PCR based sequence specific primer amplification. HLA allele and haplotype incidence in patients' group were compared to healthy population of 286 ethnically matched (Caucasian, Polish) controls. DRB1*07 allele was found in 2/4 patients, seems than to be elevated than in general Polish population but a small number of patients does not allow to perform relevant statistical tests. Moreover we found significantly more frequent B*08-DRB1*03 fragment of ancestral HLA 8.1 haplotype (8.1 AH) in PPBCL patients (3/4) than in control group (47/286) (OR=15.3; 95% CI 1.55–150; p=0.02). Peripheral blood mononuclear cells from all PPBCL patients were also studied for monoclonality in immunoglobulin (IGH VH-JH, incomplete DH-JH, IGK, IGL) and crosslineage TCR (TCRB, TCRG, TCRD) genes rearrangements according to BIOMED-2 protocol. The multiplex PCR reactions were followed with heteroduplex analysis of PCR products. We found polyclonality of lymphocytes population in all tests except incomplete DH7-JH IGH rearrangements in 2 patients. The described gene rearrangement was not related to other upstream DH or downstream JH gene segments. The HLA incidence we found is distinct from previously described elevated incidence HLA DRB1*07 allele and may suggest another genetic background of PPBCL pathogenesis. Moreover, our finding of high prevalence of 8.1 AH, known to be more frequent in autoimmune diseases, and to worsen the prognosis in some infectious diseases and non-Hodgkin lymphomas, as well as of the incomplete DJ monoclonality in 2/4 patients, warrant further studies aiming to determine the relationship between PPBCL, immune disorders and true lymphoproliferative diseases.