Absolute Lymphocytosis Research Articles

DISTINCT CLINICOLABORATORY CHARACTERISTICS of the t(14;19)(q32;q13)-POSITIVE SPLENIC MARGINAL ZONE LYMPHOMA

Abstract 5200 Background:Splenic marginal zone lymphoma (SMZL) is a well recognized B-cell neoplasm which is characterized by splenomegaly, bone marrow involvement, immunologically by typical phenotype of marginal zone cells. The most frequent cytogenetic findings are involvement of chromosomes 1, 3, 7(usually deleted in 7q) and 8. The t(14;19)(q32;q13) is a rare cytogenetic abnormality with bcl-3 rearrangement that has been reported in other B-cell lymphomas. Aims:To describe the clinical, morphological, immunophenotypic findings in SMZL associated with t(14;19)(q32;q13). Methods and results:In Hematological Research Centre, Moscow between January 2001 and May 2011 three cases SMZL with t(14;19)(q32;q13) were identified. All patients were males with age 51, 58, 67 y.o. Lymphoma presented with B-symptoms, high level of lactate dehydrogenase (LDH), hepatosplenomegaly and regional lymphadenopathy (enlarge splenic hilar lymphnodes). The hemoglobin was 92 g/l, 110 g/l, 122 g/l. All patients had normal count of leukocytes with an absolute lymphocytosis (lymphocytes count 72 × 109 g/l, 79 × 109 g/l, 83 × 109/l) and thrombocytopenia. Morphological examination of peripheral blood and bone marrow lymphocytes showed that all lymphocytes are atypical with wide cytoplasm and nuclear indentation. In all cases there was nodular type of bone marrow involvement, composed of majority medium sized cells. Immunophenotypic analysis has shown the expression of mature B-cells antigens (CD19, CD20, CD22, FMC7, sIg) and absence of ÑD10, CD23, CD5, CD43, CyclinD1.Two patients were treated with CHOP-regimen without any response. They progressed with spleen enlargement and decreased of thrombocytes counts. All 3 patients undergo splenectomy. Weight of spleen was 1800 g, 2083 g, 2850 g. Splenic section generally show massive nodular pattern (involvement of the white and red pulp) associated with diffuse invasion of the sinuses. In all cases discovered high Ki-67. All patients demonstrated progression after splenectomy during 3–6 months that was characterized by increase of leukocytes count (range 45,4 – 101,8 × 109 /l), high level of LDH, appearance of peripheral and visceral lymph nodes. Considering the increase leucocytes, presence of lymphadenopathy in all cases CHOP, FMC regimen were used. All patients died of disease progression and infectious complications. Time of observation was 21, 30, 34 months. Conclusions:The t(14;19)(q32;q13)-positive SMZL is distinct variant which is characterized by rapid progression after splenectomy, poor responses to chemotherapy and short survival. So t(14;19)(q32;q13) may be regarded as a poor prognostic factor. Disclosures:No relevant conflicts of interest to declare.

Clonal Dasatinib Large Granular Expansion Is Associated with Suboptimal and Optimal Leukemia Net Response Criteria in Chronic Myelogenous Leukemia

Abstract 1696 Introduction:Dasatinib a BCR/ABL and Src family tyrosine kinase inhibitor (TKI) used in the treatment of Chronic Myelogenous Leukemia (CML) has recently been associated with large granular (LG) expansion. Its clinical meaning and pathogenic mechanisms are still to be elucidated. Methodology: Based on the knowledge that both LG subsets (NK and T) have cytotoxic properties implicated in anti-tumoral and immune mediated activity, we conducted a prospective study from December 2009 to August 2011 with CML patients to evaluate the clinical significance of LG lymphocytosis (LGL) and T-LG clonality in dasatinib users and characterize if these conditions are specific for dasatinib when compared to imatinib. Imatinib patients (N=28) were vertically studied as a control group. Blood samples were analyzed by complete blood count, immunophenotyping with four-color flow cytometer and RT-PCR to detect T-receptor clonality. Dasatinib group (N=36) was prospectively followed for the same parameters and also evaluated for clinical response (according to the European LeukemiaNet 2009) and side effects. Results: The mean diagnosis time for the imatinib group was 6.2 years (0.3–17.4) and 5.9 years (1.2–18.5) for the dasatinib group. In both, the mean time since beginning current TKI therapy was over than 12 months (4.6 years on imatinib, 1.7 years on dasatinib). The incidence of LGL was higher in the dasatinib group compared to the imatinib group (86% × 53.5%, p=0.005) (table 1). T-LG clonality was statically associated with response by LeukemiaNet definition (p=0.0246) (table 2). It is worth to emphasize that one patient with oligoclonal LGL had major molecular response with just 50mg of dasatinib (dose reduction because of thrombocytopenia). LGL above 800/mm3 was statically associated with clonality (p=0,0224). Excluding hematologic toxicity, significant toxicity was observed in 30% (N=11) (colitis, pleural effusion, hepatic and pancreatic toxicity) of the dasatinib users (table 3). In these patients LGL was observed in 81.8% and clonality in 63.6%. Development of hemorrhagic colitis happened in the oligoclonal patient with the highest LG peak (NK=4224/mm3 and T-LG lymphocyte =1672/mm3).Table 1:Lymphocyte profile of patients treated with imatinib and dasatinibImatinib (n=28)Dasatinib (n=36)PAbsolute lymphocytosis (>3400/mm3)0%14 (39%)0.0001Media of Absolute lymphocytosis/mm31708 (800–2700)3055 (1100–9300)–T/ NK LGL (>400/ mm3)15 (53.5%)31 (86%)0.005Media of LGL (>400/ mm3)663 (440–1080)1427 (484–3280)**–Clonal lymphocyte population*6 (21%)17 (47%)0.04Monoclonality0%23.5%–Oligoclonality100%76.5%–*Oligo/monoclonal by RT-PCR.**Media of median LGL/patient.Table 2:Response and Clonality in dasatib patientsLeukemiaNet 2009 definitionResponse* (N=31)Failure (N=5)PLGL87%80%p=0.6746NK26.9%0%T-LG26.9%50%NK+T-LG46.2%50%Clonality54.8%0%p=0.0246Oligoclonal76.5%Monoclonal23.5%*Suboptimal/optimal responseTable 3:Clinical features of dasatinib patients and T-lymphocyte clonality statusClonal (n = 17)Policlonal (n = 19)PChronic phase14 (82%)11 (58%)0.21Hematological toxicity5 (29%)4 (21%)0.70Non-hematological toxicity7 (41%)4 (21%)0.28Median dose of dasatinib125mg/d (M)108mg/d—104mg/d (O)O=oligoclonal. M=monoclonal. Discussion:Previous articles suggest that T-LG lymphocytes expressed mainly two Src kinase family members, Fyn and Lck and that inhibition of this pathway (a feature of dasatinib) interfered with their development which goes against what we find in current practice. In someway dasatinib is capable of stimulating our innate defense and leads to a selection of a T-LG clone that is associated with a better response. Conclusion: We found an important association between absolute lymphocytosis, LGL and clonality in dasatinib group that was not evident with imatinib. We were able to demonstrate that LGL above 800/mm3 was associated with T-LG clonality and that clonality was statistically associated with response by LeukemiaNet criteria for CML. This suggests that clonal T-LG expansion has a role in improving dasatinib ITK function, a feature not observed with imatinib. Perspective: If dasatinib is able to stimulate our innate anti-tumoral defenses its use should be evaluated in other kind of tumors and the molecular way it occurs should be investigated. Disclosures:Off Label Use: Dasatinib dose reduction due to toxicity.

Clinical Relevance of Immunophenotypic Characteristics in Chronic Lymphocytic Leukemia

Abstract 4604 Background:Chronic lymphocytic leukemia (CLL) has the highest incidence and prevalence of any leukemia in the United States. It has a characteristic immunophenotype, but variant forms (typically termed “atypical CLL”) are commonly encountered. It is not known if this immunophenotypic heterogeneity in CLL is clinically relevant. We hypothesized that the immunophenotype of CLL cells correlates with both biologic parameters and clinical outcomes. Methods:We performed a retrospective review of clinical, biologic, and flow cytometric data of patients undergoing an evaluation of an absolute lymphocytosis at the Duke University and Durham VA Medical Centers. Patients meeting immunophenotypic criteria for CLL based on cell surface expression of CD5, CD19, CD20, CD23, and surface immunoglobulin light chain (sIg) were included in this analysis. Atypical CLL was defined if any of the following were observed: (1) expression of CD5, CD20, CD23, or sIg that differed from a typical CLL immunophenotype (e.g. bright CD20 or low CD23; hereafter non-standard immunophenotype), (2) expression of T cell markers CD7 or CD8, or (3) expression of CD123. Through electronic chart review, clinical and biologic parameters were abstracted. These included age; gender; ethnicity; WBC, hemoglobin, platelet count, and Rai stage at diagnosis; time to first treatment; overall survival; CD38, ZAP70, IGHV mutation status, and interphase cytogenetics (FISH). A chi square test or Fisher's exact test for small sample sizes was used to test association of categorical data. The central tendencies of continuous measurement were compared using the Wilcoxon rank sum test. Overall survival and time to treatment were calculated using the Kaplan-Meier product limit method. Survival curves were compared using the log-rank test. This study was approved by IRBs at Duke University and Durham VA Medical Centers. Results:We reviewed 189 patients, including 119 (63%) with typical CLL immunophenotype and 70 (37%) with atypical. Among patients with atypical CLL, 93% (65 of 70) had a non-standard immunophenotype, 33% (23 of 70) had expression of T cell markers, and 33% (14 of 43) had expression of CD123. We observed expression of T cell markers in 12% (23 of 189) and CD123 in 12% (14 of 114) of the entire cohort, respectively. At the time of diagnosis, there was no association between typical or atypical CLL immunophenotype and age, gender, race/ethnicity, WBC, hemoglobin, platelet count, or Rai stage (p>0.05 for all comparisons). With regard to biologic parameters, there was no association of CLL immunophenotype with the proportion of patients with CD38 ≥30%; however, the central tendency of CD38 was higher in the atypical group when analyzed as a continuous variable (median value 38% vs. 10%, p=0.04). There was no difference in the proportion of ZAP70 positive cases between typical and atypical groups. Analysis of interphase cytogenetics (FISH) data showed that a higher percentage of typical than atypical immunophenotypes had del 13q as a sole abnormality and a lower percentage had trisomy 12 (p=0.02). Despite the increased frequency of del 13q as a sole abnormality, lower frequency of trisomy 12, and lower expression of CD38, there was no difference between groups in time to first treatment (median 5.3 years for typical vs. 2.1 years for atypical; p= 0.11) or in overall survival (median 13.6 yrs64 years for typical vs. 18.0 years for atypical; p=0.11) (Figure 1). However, when patients with non-standard CLL immunophenotype were compared to those with a standard immunophenotype, a significantly shorter time to first treatment was observed (median 6.0 years for standard immunophenotype vs. 2.2 years for non-standard, p=0.03). [Display omitted] Conclusion:Different immunophenotypic subtypes are commonly encountered in the care of patients with CLL. These groups have different biological characteristics, including differences in expression of CD38 and cytogenetic abnormalities. Exploratory analysis also showed differences in clinical outcomes based on immunophenotypic parameters. Validation studies are currently underway. Disclosures:No relevant conflicts of interest to declare.

Clinical, Phenotypic, and Genetic Characteristics of Non-CLL Type Monoclonal B Lymphocytosis (MBL)

Abstract 5209Monoclonal B-cell lymphocytosis (MBL) has been established as a diagnostic category to denote the finding of clonal B lymphocytes in otherwise healthy and asymptomatic individuals. MBL likely represents early lesions in lymphoid neoplasms. Immunophenotypically, MBL is divided into CD5+ CLL-type and CD5- non-CLL type MBL, respectively. While most studies have focused on CLL-type MBL and provided evidence of progression of MBL to clinical CLL, the biological nature and clinical relevance of non-CLL type MBL remain unclear. In this study, we have identified and evaluated a series of 18 patients with non-CLL type MBL. This group comprised 10 males and 8 females with a median age of 74 years (range 53–95). All the patients presented initially with a mild to moderate absolute lymphocytosis. Clinical and laboratory evaluations were performed with a follow-up period of 1–20 years (median 8.5). Clonal B lymphocyte populations were confirmed by flow cytometry analysis. All the MBL clones showed negative for CD5, CD10 and CD103, positive for CD19 and CD20, and heterogeneous for CD23, CD25, CD79b, FMC7 and CD38. The phenotypic findings excluded CLL, mantle cell lymphoma and hairy cell leukemia, but may overlap with marginal zone lymphoma or lymphoplasmacytic lymphoma. Cytogenetics were available for 9 patients. Of the cytogenetic abnormalities, 7q deletion was found in two clones, 17 q deletion in two other clones, 11q deletion in one clone and t(2;7) in another clone. An overall tendency was shown toward clonal expansion with an increase in absolute lymphocyte count (ALC) over time (initial mean ALC of 4.9 ± 0.46 × 109/L to a recent mean ALC of 8.3 ± 2.18 × 109/L, p = 0.151). Three patients had an initial ALC >8 × 109/L which remained stable in one patient and decreased to <6 × 109/L in 2 patients. The patient with the longest follow-up of 20 years underwent a completely benign course, while the CD5- clonal B cell population was persistent but non-progressing. Of the progressive clones, three patients had an increase in ALC >10 × 109/L; one patient’s ALC progressed over a period of 9 years to 35 × 109/L that required treatment. Clinically, three patients developed splenomegaly and one patient progressed with lymphadenopathy. Unlike the findings from population screening, this study represents a series of clinical MBL of non-CLL phenotype, and our results indicate the heterogeneity in genetic changes and clonal progression. Disclosures:No relevant conflicts of interest to declare.

Case study interpretation—Houston: Case 1

Case study interpretation—Houston: Case 1

Fatal Paraneoplastic Systemic Leukocytoclastic Vasculitis as a Presenting Feature of Chronic Lymphocytic Leukemia

The most common paraneoplastic vasculitis is leukocytoclastic vasculitis (LCV),(1) 75% of which are caused by hematological malignancies. Chronic lymphocytic leukemia (CLL) is associated with a multitude of auto-immune paraneoplastic syndromes. Data on LCV in association with CLL is restricted to isolated case reports,(3,4) none of which had systemic LCV. We present a unique case of fatal paraneoplastic, systemic LCV as an initial presentation of CLL in an elderly male with multiple co-morbidities. A 71-year-old man presented with a palpable, symmetric, purpuric rash on the lower extremities and an absolute lymphocytosis (white blood cell count 26.9; 23% lymphocytes). His co-morbidities included coronary artery disease, congestive heart failure, and new critical aortic stenosis. Flow cytometry of peripheral blood demonstrated an abnormal population of B-cells, positive for CD5, CD19, and CD23, consistent with CLL. The skin biopsy specimen revealed neutrophilic inflammation in vessel walls indicative of LCV. Acute renal failure (creatinine 2 mg/dL), urinary red cell casts, and hypocomplementemia were concerning for a systemic vasculitis. The antinuclear antibody, cryoglobulin titer, antineutrophil cytoplasmic antibody, serum protein electrophoresis, viral serologies were negative. On hospital day 6, he developed acute hepatocellular injury and acute respiratory failure. Continuous veno-venous hemodialysis was begun for worsening acidemia and hyperkalemia. Two days later he became obtunded on hospital day 8 and had an elevated lactic acid level with generalized abdominal tenderness worrisome for bowel ischemia. The same day he needed intubation with cardiopulmonary resuscitation for a brief episode of asystole. Despite aggressive treatment with high-dose steroids and plasmapheresis, he suffered worsening renal failure and shock. His family sought withdrawal of care on hospital day 11. Autopsy revealed diffuse LCV of the stomach, distal ileum, integument and alveoli with petechial hemorrhages, fibrin thrombi, and gangrenous patchy necrosis. Paraneoplastic LCV is a rare syndrome and seldom occurs in association with CLL. This is the first reported case of fatal systemic paraneoplastic LCV from B-cell CLL. Dermatologic involvement is universal with LCV, and may portend systemic disease. More data on its pathogenesis in CLL is warranted.

Cutaneous Papules, Hypercalcemia, and Osteolytic Bone Lesions

A 61-year-old Japanese American woman was hospitalized with recalcitrant hypercalcemia of 3 months’ duration. Physical examination showed generalized cutaneous papules. The patient also had osteolytic bone lesions that caused fractures. On admission, the calcium level was 17.5 mg/dL (to convert to mmol/L, multiply by 0.25), and the parathyroid hormone level was suppressed. Peripheral blood smear showed a slight absolute lymphocytosis with occasional atypical lymphocytes. A skin biopsy specimen revealed a dense dermal infiltrate of atypical T lymphocytes with strong CD25 expression, characteristic of human T-cell lymphotropic virus type 1 (HTLV-1)–associated adult T-cell leukemia/lymphoma (ATCLL). Serologic testing for HTLV-1 was subsequently shown to be positive. The HTLV-1 is endemic in Japan, the Caribbean, Central Africa, and the southeastern United States. Infection is transmitted by sexual contact, blood transfusion, or mother-to-child vertical transmission. The HTLV-1 genome is clonally integrated into the DNA of the lymphoma cells. The clinical differential diagnosis of the skin findings in this patient included generalized histiocytic disorders, leukemia cutis, and other types of lymphoma. The diagnosis is established through identification of the leukemia/lymphoma cells within the peripheral blood, skin, or other affected organs. The strong expression of CD25 by the lymphoma cells is a characteristic immunophenotypic finding. With conventional systemic chemotherapy regimens, the prognosis for patients with ATCLL is poor. Recent studies have examined other potentially more promising treatments, such as allogeneic stem cell transplant, alemtuzumab, denileukin diftitox, or antiviral agents. Although rare in the United States, ATCLL should be considered in patients from endemic areas who present with cutaneous lymphoid infiltrates, hypercalcemia, and osteolytic bone lesions.

Subclinical chronic lymphocytic leukemia with atypical cutaneous presentation

Chronic lymphocytic leukemia (CLL) involving skin is a rare but well-documented occurrence, mainly reported in advanced disease. In contrast, CLL presenting with skin lesions is exceedingly rare, only few reports existing to date. We report a 70-year-old man who presented with two non-pruritic, papular lesions on the lower abdomen and proximal thigh. Biopsies showed dense lymphohistiocytic infiltrates involving the reticular dermis and subcutis without epidermotropism consisting mostly of small, CD20 and PAX-5-positive B-cells expressing CD5, CD23, CD43 and BCL2. Numerous large B-cells were present in a T-cell, histiocyte-rich background. A staging bone marrow biopsy showed a clonal B-cell proliferation with typical CLL flow cytometry immunophenotyping but neither lymphadenopathy nor absolute lymphocytosis was present. Numerous B and T-cell cutaneous lymphoproliferative disorders can be associated with increased numbers of histiocytes occasionally masquerading as benign disorders. This was the case with our patient's lesions, originally interpreted as cutaneous Rosai-Dorfman disease. A high index of suspicion from both the pathologist and the dermatologist is essential in identifying these rare but probably underrecognized occurrences of early systemic lymphoproliferative disorders presenting as cutaneous lesions with an unexpected cellular composition.

Evidence-Based Mini-Review: The Role of Alkylating Agents in the Initial Treatment of Chronic Lymphocytic Leukemia Patients with the 11q Deletion

A 55-year-old man presented with fever, night sweats, and weight loss of about 20 lbs. in the prior 6 months. Physical examination revealed multiple cervical, axillary, and inguinal lymphadenopathy. The spleen was enlarged. A complete blood count revealed leukocytosis with absolute lymphocytosis: 30,000/μL. Peripheral blood-flow cytometric analysis showed a clonal lymphocyte population with immunophenotypes positive for CD5, CD20(dim), and monotypic kappa light chain. Fluorescence in situ hybridization (FISH) analysis revealed del(11q22.3), but negative for t(11:14). What should be used to treat his chronic lymphocytic leukemia (CLL) disease?

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

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

Higher Bone Marrow Lymphocyte Percentage 3 to 6 Month After Therapy with Hypercvad + Dasatinib Than That with Hypercvad + Imatinib or Hypercvad Alone In Patients with Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

AbstractAbstract 4336 Background:Recent reports have suggested that aside from the tyrosine kinase inhibitory effect, dasatinib also promotes a clonal expansion of cytotoxic T-cells in patients with Philadelphia-chromosome positive (Ph+) leukemias. We sought to determine whether there is an absolute lymphocytosis in patients with Ph+ Acute Lymphocytic Leukemia (ALL) treated at our institution on 3 consecutive protocols. Method:We collected data on 122 patients with Ph+ ALL treated with 3 different regimens (hyperCVAD, hyperCVAD + imatinib, and hyperCVAD + dasatinib) from 1994 to 2010, who achieved a complete hematological response (CHR). We examined potential differences between the absolute numbers lymphocytes in peripheral blood (PB) and percentage lymphocytes in bone marrow (BM) at 3 stages: pretreatment, at CHR, and 3–6 months after CHR. Result:Thirty four (28%) patients received hyperCVAD, 43 (35%) hyperCVAD + imatinib, and 45 (37%) hyperCVAD + dasatinib. Twenty five (74%) of patients treated with hyperCVAD, 14 (33%) of patients treated with hyperCVAD + imatinib, and 5 (11%) of patients treated with hyperCVAD + dasatinib have relapsed on these sequential studies. The median PB absolute lymphocyte count for the 3 groups are as follow: Pretreatment: hyperCVAD: 3570.5/uL, hyperCVAD + imatinib: 2262/uL, hyperCVAD + dasatinib: 2376/uL (p = 0.1); at CR: hyperCVAD: 291.5/uL, hyperCVAD + imatinib: 168/uL, hyperCVAD + dasatinib: 239/uL (p = 0.3); 3 to 6 months after CR: hyperCVAD: 485/uL, hyperCVAD + imatinib: 450/uL, hyperCVAD + dasatinib: 632.5/uL (p = 0.26). The median BM lymphocyte percentages are as follow: Pretreatment: hyperCVAD: 3%, hyperCVAD + imatinib: 6%, hyperCVAD + dasatinib: 5% (p = 0.5). CHR: hyperCVAD: 6%, hyperCVAD + imatinib: 4%, hyperCVAD + dasatinib: 4% (p = 0.4). Three to 6 months after CHR: hyperCVAD: 5%, hyperCVAD + imatinib: 3%, hyperCVAD + dasatinib: 7% (p = 0.005). We then divided all patients by their 3 to 6 month BM lymphocyte percentages to 3 groups (< 3%, 3–6%, >6%). There was no difference in remission-free survival for these 3 groups (p=0.76)(Figure 1) Conclusion:Patients who received hyperCVAD + dasatinib had a statistically significant higher BM lymphocyte percentages, 3 to 6 month after achieving CHR than patients treated with the imatinib-containing regimen or chemotherapy alone. Whether this is a clonal population of effector T-cells and whether it plays an important role on the activity of dasatinib compared to imatinib or chemotherapy alone will need to be further investigated. Disclosures:Ravandi:Novartis: Honoraria, Speakers Bureau; Bristol- Myere- Squibb: Honoraria, Research Funding. Kantarjian:BMS, Pfizer and Novartis: Research Funding; Novartis: Consultancy. Cortes:Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Pfizer: Consultancy, Research Funding. O'Brien:Novartis: Research Funding; BMS: Research Funding. Thomas:Novartis: Honoraria; Bristol-Meyer-Squibb: Honoraria; Amgen: Research Funding. [Display omitted]

Use of the CD19 count in a primary care laboratory as a screening method for B-cell chronic lymphoproliferative disorders in asymptomatic patients with lymphocytosis

Detection of absolute and relative lymphocytosis in otherwise asymptomatic elderly patients is very common in the primary care setting and frequently results in referral for screening of lymphoproliferative disorders. Since many B-cell chronic lymphoproliferative disorders (B-CLPD) are indeed asymptomatic at diagnosis in most patients with lymphocytosis, no sign of such a disorder is usually detected. Currently, specific guidelines for screening of patients with lymphocytosis are lacking. We investigated the practicability and clinical value of a single colour CD19 count performed by a primary care laboratory in order to improve the diagnostic follow-up of patients with lymphocytosis in a primary care laboratory. The capability of detecting monoclonal B-cell lymphocytosis and B-CLPD by CD19, was first confirmed in patient samples with known B-CLPD. Next, in a previously defined geographic area, a CD19 count was performed on all samples for patients aged ≥40 years with relative or absolute lymphocytosis but without neutropenia. Clinical follow-up, with a median of 4 years, was performed using both a survey among the requesting general practitioners and by analysis of the records of the referral hospitals within the borders of the defined area. A total of 520 cases with asymptomatic lymphocytosis were identified. In all cases, the CD19 count was performed; 207 (40%) showed increased values and 313 (60%) showed normal values. An increase in CD19 proved highly sensitive for detection of B-CLPD (98%, 95% CI; 94%-100%) with a high positive predictive value (57%, 95% CI; 50%-63%). The area under curve, the receiver-operating characteristic curve of the CD19 count (0.93, 95% CI; 0.91-0.96), was significantly higher compared to the absolute lymphocyte count (0.86, 95% CI; 0.83-0.89), especially in patients with moderate lymphocytosis. This study indicates that the CD19 count, performed by a primary care laboratory, is feasible and a promising tool for initial screening of lymphocytosis to discriminate B-CLPD from benign causes of lymphocytosis.

Mono/oligoclonal T and NK cells are common in chronic myeloid leukemia patients at diagnosis and expand during dasatinib therapy

In a proportion of patients with chronic myeloid leukemia (CML) being treated with dasatinib, we recently observed large granular lymphocyte (LGL) expansions carrying clonal T-cell receptor (TCR) gamma/delta gene rearrangements. To assess the prevalence and role of clonal lymphocytes in CML, we collected samples from patients (n = 34) at the time of diagnosis and during imatinib and dasatinib therapies and analyzed lymphocyte clonality with a sensitive polymerase chain reaction-based method of TCR gamma and delta genes. Surprisingly, at CML diagnosis, 15 of 18 patients (83%) had a sizeable clonal, BCR-ABL1 negative lymphocyte population, which was uncommon in healthy persons (1 of 12; 8%). The same clone persisted at low levels in most imatinib-treated patients. In contrast, in a distinct population of dasatinib-treated patients, the diagnostic phase clone markedly expanded, resulting in absolute lymphocytosis in blood. Most patients with LGL expansions (90%) had TCR delta rearrangements, which were uncommon in patients without an LGL expansion (10%). The TCR delta clones were confined to gammadelta(+) T- or natural killer-cell compartments and the TCR gamma clones to CD4(+)/CD8(+) alphabeta(+) fractions. The functional importance of clonal lymphocytes as a part of leukemia immune surveillance and the putative anergy-reversing role of dasatinib require further evaluation.

Cytomegalovirus presenting with abdominal pain and pyrexia of unknown origin

Dear EditorCytomegalovirus (CMV) is a member of the herpesvirus family with ubiquitous transmission. Infection is mostoften asymptomatic, with exceptions being the immuno-compromised and those in utero. Like other members ofthe herpesvirus family, CMV establishes latent infectionafter the resolution of acute infection. Seroprevalence ratesvary between 40 and 60%, increasing with age and areinversely proportional to socioeconomic status. A syn-drome resembling infectious mononucleosis is the mostcommon presentation of symptomatic CMV infection inimmunocompetent adults. Classic mononucleosis is anillness characterised by significant, often protracted feversand lassitude in the setting of absolute lymphocytosis withatypical lymphocytes. The mononucleosis syndrome asso-ciated with CMV infection has been described as‘‘typhoidal’’ in presentation, since systemic symptoms andfever predominate and signs of enlarged cervical nodes andsplenomegaly are not as commonly seen as they are inEpstein Barr Virus (EBV) [1]. Here, we present the case ofan acute CMV infection that presented as a surgicaladmission of abdominal pain with pyrexia.A 54-year-old Irish Caucasian farmer was referred to theaccident and emergency department by his general practi-tioner for an 18-day history of left iliac fossa (LIF) pain.He had a background history of a total colectomy forhereditary nonpolyposis colorectal cancer (HNPCC)3 years prior and a left-sided orchidectomy as a child. TheLIF pain was rated as mild to moderate in severity andintermittently crampy in nature. Radiation to his back wasnoted along with associated night sweats. The patientdenied any history of changes in bowel habit, blood perrectum, anorexia or weight loss.On examination, he was apyrexial with normal respira-tory parameters, blood pressure 150/80 mmHg and a sinustachycardia at 120 beats/min. Cardiovascular, respiratoryand neurological examinations were all unremarkable.Abdominal examination revealed a soft non-tender abdo-men with no masses or organomegaly. Bowel sounds werepresent and PR examination was unremarkable with anegative test for faecal occult blood. Preliminary investi-gations showed normal haematology and biochemistry,with the exception of an elevated C-reactive protein (CRP)at 43 mg/l. Urinary dipstick was unremarkable, as was thepresenting chest X-ray (CXR). Blood cultures and MSUwere taken.

First‐line therapy with doxycycline in ocular adnexal mucosa‐associated lymphoid tissue lymphoma: A retrospective analysis of clinical predictors

This retrospective study was launched to evaluate the efficacy of doxycycline and to find independent predictors of a clinical response in patients with ocular adnexal lymphoma of mucosa-associated lymphoid tissue (OAML). Thirty-eight patients with newly diagnosed, localized OAML received doxycycline for 3 weeks (12 patients) or 6 weeks (26 patients). Clinical factors including absolute lymphocyte count (ALC) and neutrophil count (ANC) were compared between responders and non-responders. After a median follow-up of 26.4 months, doxycycline resulted in an overall response rate of 47% and a 3-year time-to-treatment failure (TTF) rate of 84%. Patients treated with doxycycline for 6 weeks versus 3 weeks tended to have a higher response rate (54%vs 33%). Absolute lymphocytosis (ALC > 3.01 x 10(9)/L) and absolute neutrophilia (ANC > 1.92 x 10(9)/L) were defined based on the median value of each count. Patients with (19 patients) versus without absolute lymphocytosis had significantly shorter 2-year TTF (70%vs 100%, P = 0.021) and a lower response rate (32%vs 63%, P = 0.051). Absolute lymphocytosis (odds ratio [OR] = 4.7; 95% confidence interval [CI], 1.1-20.8; P = 0.043) and non-conjunctival tumor (OR = 11.8; 95% CI, 1.1-122.5; P = 0.038) were negative predictors for response by multivariate analysis. Front-line doxycycline is effective particularly in localized OAML patients without absolute lymphocytosis but with conjunctival involvement.

Soluble CD138 serum levels are not associated with other poor prognostic markers in patients with B-chronic lymphocytic leukaemia

The clinical course of CLL is highly variable, and survival from the time of diagnosis of CLL can range from months to decades. Novel biological markers such as IgVH mutation, CD38, and ZAP-70 expression have shown to offer important prognostic informations. Few reports deal with the sCD138 levels and bad prognostic factors in patients with CLL, and contrasting data are reported in literature. In our study, we evaluated the serum level of sCD138 in patients with B-CLL and its relationship with other prognostic markers. There was a significant association between advanced Rai stage and serum sCD138 levels in CLL subjects. Patients with Rai stage III-IV had significantly higher levels of sCD138 with respect to controls (48.85±34 ng/ml vs. 31.1±19.34 ng/ml; P<0.05). We were unable to demonstrate a significant association between sCD138 serum levels and IgVH gene status, ZAP-70 expression, CD38 expression, beta-2 microglobulin, absolute peripheral blood lymphocytosis, haemoglobin or LDH levels. Our finding that high sCD138 serum levels correlates with advanced stages in patients with B-CLL is consistent with the possibility molecule can identify patients with high tumour burden, but the lack of correlation between sCD138 serum levels and markers such the mutation status of IgVH, ZAP-70, and CD38 suggests that sCD138 levels only reflect the clinical stage of disease than the clinical course or progression.

SINAIS NERVOSOS E MORTE ASSOCIADOS A ACARICIDA CONTAMINADO POR TORDON® (2,4 - D, 64/240 TRIETANOLAMINA) EM BEZERROS, NO MUNÍCIPIO DE GUARAPARI, NO ESPÍRITO SANTO, BRASIL

This study investigates a type of neurological syndrome in two calves. They were sprayed with high cis-cypermethrin and chlorpyrifos after the same pump sprayer had been used to apply tordon herbicide to pasture. Clinical findings revealed compulsive licking of hair coat, anorexia, diarrhea, dyspnea, depression, collapse, pale mucous membranes, dehydration, hyperthermia, rotation of the eyeball, third eyelid protrusion, enophthalmos, ventromedial strabismus, costo-abdominal breathing with increased expiratory effort, drooling, ventral position of head during walk, mild incoordination, impaired proprioception related to thorax and pelvis rotation, miosis, as well as absence of consensual and bilateral pupillary reflex, suggesting lesion of the mesencephalon. Laboratory findings indicated reduction in MCHC, absolute lymphocytosis, relative and absolute monocytosis; on the other hand, urea, creatinine, AST, alkaline phosphatase and GGT levels increased. As regards pathological findings, there was evidence of cerebral congestion and neuronal necrosis, hematoma in rete mirabile, multifocal necrosis and hemorrhage in liver and kidneys, multifocal, coagulative necrosis of the renal tubular epithelium, hyaline degeneration, and hypertrophic nuclei. One of the calves showed severe secondary bronchopneumonia with fibrous adhesions between the pleural and pericardial sac. The lesions observed in these cases should not be attributed to the chemicals' individual reactions. It is the combination of acaricide with tordon that may have enhanced each substance's toxicity, leading to poisoning and killing of the calves.

TRATAMENTO HEPATOPROTETOR FAVORECE A RESPOSTA LEUCOCITÁRIA DE RATOS WISTAR INTOXICADOS POR CCl4

A presente investigação teve por objetivo avaliar o efeito hepatoprotetor de tratamentos com o composto deN- Acetil DL- Metionina (5%) + Cloreto de Colina (2%) + Cafeína (1%) + Tiamina Cloridrato (1%) +Nicotinamida (0,5%) + Piridoxina Cloridrato (0,04%), administrado via Intramuscular (IM) nas doses de 0,2;0,6 e 1,0 L/Kg de peso vivo (PV) , através do estudo leucocitário em ratos Wistar, submetidos à intoxicaçãoaguda com CCl4. Fêmeas (N=147) foram distribuídas aleatoriamente em 21 grupos, constituindo seis diferentes tratamentos, sendo avaliados sete animais por grupo em quatro períodos: dois, quatro, seis e oitodias pós-intoxicação induzida pelo CCl4. Verificou-se neste estudo quadros de eosinofilia e monocitose absoluta em animais não tratados e tratados com a dose mínima de 0,2 mL, sendo estes efeitossignificativamente amenizados em animais tratados com 0,6 e 1,0 mLdo composto hepatoprotetor/kg de PV. Os animais intoxicados e não tratados apresentaram um quadro detrombocitopenia, quando comparado a animais intoxicados e tratados. Houve neutropenia e linfocitoseabsoluta em todos os ratos intoxicados com CCl4, não existindo diferença entre os tratamentos. A análise do leucograma demonstrou que o tratamento com o composto hepatoprotetor favoreceu a respostaleucocitária, por atuar beneficamente sobre a população destas células, corroborando a hipótese de que taiseventos amenizem os efeitos deletérios da intoxicação por CCl4 no tecido hepático.

Chronic lymphocytic leukemia With t(2;14)(p16;q32) involves the BCL11A and IgH genes and is associated with atypical morphologic features and unmutated IgVH genes.

The t(2;14)(p16;q32) has been reported previously in only 12 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). The clinicopathologic features have been incompletely described. We describe 6 new cases of CLL/SLL with t(2;14)(p16;q32). All had marrow involvement, 4 had absolute lymphocytosis, 4 had lymphadenopathy, and 3 had hepatosplenomegaly. All showed atypical lymphocyte morphologic features with plasmacytoid differentiation and irregular nuclei; 3 had increased prolymphocytes. Flow cytometry demonstrated typical immunophenotypes in 5 and an atypical immunophenotype in 1. All expressed ZAP70; 5 assessed showed unmutated IgV(H) genes. Karyotyping identified t(2;14)(p16;q32) as the sole abnormality in 1, primary abnormality in 2, and part of a complex karyotype in 3. Fluorescence in situ hybridization analysis revealed BCL11A/IgH rearrangement in all. After chemotherapy, 3 patients died of disease and 3 were alive with disease (median follow-up, 80 months). We conclude that CLL/SLL with t(2;14) (p16;q32) and BCL11A/IgH rearrangement is characterized by atypical morphologic features and unmutated IgV(H) genes.

Facteurs pronostiques utiles dans l’évaluation personnalisée d’un patient avec une leucémie lymphoïde chronique (LLC)

B cell chronic lymphocytic leukaemia (B-CLL) is the most frequent leukaemia, particularly in older patients. B-CLL is defined by the presence of an absolute lymphocytosis with a RMH scoring system above or equal to 4. Clinical staging of the disease has assisted in deciding treatment options and more recently the cytogenetic (del 17p13, del 11q22-q23), molecular expression of unmutated immunoglobulin heavy chain variable region genes (IGHV), proliferation markers (thymidine kinase or CD23s) and surrogate markers (CD38, ZAP-70) have assisted in further risk stratification. A high number of other prognostic factors (LPL, ADAM) have been recently described but they are not useful in routine. The improvement of the knowledge of B-CLL is the basis of an optimal management of patients with B-CLL, including individualized treatment decisions. La leucémie lymphoïde chronique (LLC) est une hémopathie fréquente, en particulier chez le sujet âgé. Elle se définit par la présence d’une lymphocytose sanguine absolue supérieure à 4 × 109/l avec à l’examen par cytométrie en flux une prolifération monotypique et un score RMH supérieur ou égal à 4. Quatre facteurs avec un impact fort sur le pronostic ont été identifiés : temps de doublement des lymphocytes (TDL) supérieur à 12 mois, profil non muté des gènes des chaînes lourdes des immunoglobulines (IGHV), thymidine kinase ou CD23 soluble sériques élevés, présence d’une del 17p13 ou del 11q22-q23. De nombreux autres facteurs ont été plus récemment décrits mais ne sont pas utilisés en pratique quotidienne comme facteur de substitution de l’étude du profil IGHV. L’amélioration des connaissances permet une meilleure prise en charge des patients avec une LLC, prise en charge individualisée dépendante du parcours du patient.