Guidelines for Diagnosis and Treatment of Chronic Lymphocytic Leukemia. Krohem B-Cll 2017.

Recent developments in the diagnosis and treatment of chronic lymphocytic leukemia (B-CLL) have led to change of approach in clinical practice. New treatments have been approved based on the results of randomized multicenter trials for first line and for salvage therapy, and the results of numerous ongoing clinical trials are permanently providing new answers and further refining of therapeutic strategies. This is paralleled by substantial increase in understanding the disease genetics due to major advances in the next generation sequencing (NGS) technology. We define current position of the Croatian Cooperative Group for Hematologic Disease on diagnosis and treatment of CLL in the transition from chemo-immunotherapy paradigm into a new one that is based on new diagnostic stratification and unprecedented therapeutic results of B-cell receptor inhibitors (BRI) and Bcl-2 antagonists. This is a rapidly evolving field as a great number of ongoing clinical trials con-stantly accumulate and provide new knowledge. We believe that novel therapy research including genomic diagnosis is likely to offer new options that will eventually lead to time limited therapies without chemotherapy and more effective clinical care for B-CLL based on individualized precision medicine.

Randomized Phase II Trial Comparing Obinutuzumab (GA101) With Rituximab in Patients With Relapsed CD20+ Indolent B-Cell Non-Hodgkin Lymphoma: Final Analysis of the GAUSS Study.

PURPOSE: Obinutuzumab (GA101), a novel glycoengineered type II anti-CD20 monoclonal antibody, demonstrated responses in single-arm studies of patients with relapsed/refractory non-Hodgkin lymphoma. This is the first prospective, randomized study comparing safety and efficacy of obinutuzumab with rituximab in relapsed indolent lymphoma. The primary end point of this study was the overall response rate (ORR) in patients with follicular lymphoma after induction and safety in patients with indolent lymphoma. PATIENTS AND METHODS: A total of 175 patients with relapsed CD20(+) indolent lymphoma requiring therapy and with previous response to a rituximab-containing regimen were randomly assigned (1:1) to four once-per-week infusions of either obinutuzumab (1,000 mg) or rituximab (375 mg/m(2)). Patients without evidence of disease progression after induction therapy received obinutuzumab or rituximab maintenance therapy every 2 months for up to 2 years. RESULTS: Among patients with follicular lymphoma (n = 149), ORR seemed higher for obinutuzumab than rituximab (44.6% v 33.3%; P = .08). This observation was also demonstrated by a blinded independent review panel that measured a higher ORR for obinutuzumab (44.6% v 26.7%; P = .01). However, this difference did not translate into an improvement in progression-free survival. No new safety signals were observed for obinutuzumab, and the incidence of adverse events was balanced between arms, with the exception of infusion-related reactions and cough, which were higher in the obinutuzumab arm. CONCLUSION: Obinutuzumab demonstrated a higher ORR without appreciable differences in safety compared with rituximab. However, the clinical benefit of obinutuzumab in this setting remains unclear and should be evaluated within phase III trials.

[Clinical features in DLBCL and translocation BCL2/c-MYC "double hit" lymphoma]. / Klincke karakteristike u bolesnika s difuznim B velikostanicnim limfomom I dvostrukom translokacijom BCL2/c-MYC.

Diffuse large B-cell lymphoma (DLBCL) is classified as lymphoma and various entities using the gene expression of proteins are classified into three groups. The aim of this study was to clarify the clinical, biological, immunophenotypic and cytogenetic features of DLBCL with translocation t (14; 18) and 8q24/c-MYC. Eleven DLBCL patients with dual translation were monitored during the 2000-2009 period. The characteristics of these patients included morphological, immunohistochemical and cytogenetic analysis. Study results showed that all patients had aggressive characteristics, presence of B symptoms (64%), general patient condition according to ECOG scale ≥ 2 (55%), elevated serum lactate dehydrogenase activity (73%), clinical stage III and IV (82%), extranodal involvement of the disease (73%), and IPI ≥ 2 (73%). Partial remission was achieved in 73% of all patients and all patients (73%) died within a short time. Patients were treated with CHOP and similar protocols (COP, CVP, CNOP), with the addition of MabThera. Immunophenotyping was performed and determined expression of the CD20, CD3, CD10, BCL6 and MUM1 markers. The cytogenetic analysis/fluorescence in situ hybridization revealed complex karyotype changes. Thus, we analyzed the presence of BCL2, BCL6 and c-MYC genes and found eight patients to have BCL2 and c-MYC translocation genes, while three had translocation of the BCL6 and c-MYC genes. Despite appropriate therapy, the patient prognosis is poor. The median survival in these patients was 1.85 years. DLBCL with BCL2 and c-MYC rearrangement of the subgroups of lymphoma is associated with very poor survival. The presence of these two translocations has an aggressive clinical course.

Fludarabine plus alemtuzumab versus fludarabine alone in patients with previously treated chronic lymphocytic leukaemia: a randomised phase 3 trial.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is an incurable and chronic disorder, with worsening prognosis for patients as their disease progresses. We compared the efficacy and safety of the combination of fludarabine and alemtuzumab with fludarabine monotherapy in previously treated patients with relapsed or refractory CLL. METHODS: Patients (aged ≥ 18 years) with CLL Binet stage A, B, or C or Rai stages I-IV were randomly assigned in a 1:1 ratio according to a computer-generated allocation schedule to open-label combination treatment (fludarabine 30 mg/m(2) per day and alemtuzumab 30 mg per day on days 1-3) or monotherapy (fludarabine 25 mg/m(2) on days 1-5) by use of an interactive voice response system. Both regimens were given intravenously for a maximum of six 28-day cycles. The primary endpoint was progression-free survival (PFS). Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00086580. FINDINGS: Fludarabine plus alemtuzumab (n=168) resulted in better PFS than did fludarabine monotherapy (n=167; median 23·7 months [95% CI 19·2-28·4] vs 16·5 months [12·5-21·2]; hazard ratio 0·61 [95% CI 0·47-0·80]; p=0·0003) and overall survival (median not reached vs 52·9 months [40·9-not reached]; 0·65 [0·45-0·94]; p=0·021) compared with fludarabine alone. All-cause adverse events occurred in 161 (98%) of 164 patients in the combination treatment group and 149 (90%) of 165 in the fludarabine alone group. Patients in the fludarabine plus alemtuzumab group had more cytomegalovirus events (23 [14%] vs one [<1%]) and grade 1 or 2 potentially alemtuzumab infusion-related adverse reactions (102 [62%] vs 22 [13%]). Grade 3 or 4 toxicities in the combination treatment and monotherapy groups were leucopenia (121 [74%] of 164 vs 55 [34%] of 164), lymphopenia (149 [94%] of 158 vs 53 [33%] of 161), neutropenia (93 [59%] of 157 vs 110 [68%] of 161), thrombocytopenia (18 [11%] of 164 vs 27 [17%] of 163), and anaemia (14 [9%] of 163 vs 28 [17%] of 164). The incidence of serious adverse events was higher in the combination treatment group (54 [33%] of 164 vs 41 [25%] of 165); deaths due to adverse events were similar between the two groups (ten [6%] vs 12 [7%]). INTERPRETATION: The combination of fludarabine and alemtuzumab is another treatment option for patients with previously treated CLL. FUNDING: Genzyme.

[Diagnostic and prognostic significance of CD45 cell surface antigen expression in hematologic malignancies with main focus on acute leukemias]. / Dijagnosticko i prognosticko znacenje izrazaja biljega CD45 u hematoloskim zlocudnim bolestima s posebnim osvrtom na akutne leukemije.

CD45 cell surface antigen is a transmembrane protein with tyrosine phosphatase activity, expressed by all nucleated cells of hematopoietic origin, except erythrocytes and platelets. Monoclonal antibodies directed against CD45 represent irreplaceable tool in differential diagnosis of hematologic and other, non-hematologic low differentiated malignancies, primarily in cases of: extranodal lymphomas, non-hematologic malignancies with nodal or bone marrow localization or their metastases in mentioned sites. As cell surface immunophenotype marker, CD45 is of great value in differentiation of lymphoproliferative diseases subtypes. By flow cytometry, based on CD45 expression, the malignant cell population is being identified and that fact is used in, not only diagnosis, but also in detection of minimal residual disease, especially in cases of CD45 negative acute leukemias. Incidence of childhood CD45 negative acute lymphoblastic leukemias (ALL) is about 10%. Children diagnosed with low CD45 expression ALL generally have better prognosis than those with high CD45 expression, especially when cut-off value for CD45 expression is set on 90%. We have analyzed CD45 expression by flow cytometry in 28 consecutive patients diagnosed with ALL in our institution during a 5-year period. Among these patients 7.1% were CD45 negative. A positive correlation between CD45 and CD20 expression was found, and a negative correlation between CD45 and CD34. In our group of patients, CD45 expression did not have any influence on survival.

[Association of CD34 cell surface antigen expression with cytomorphological characteristics of acute promyelocytic leukemia blasts and clinical characteristics of patients: one center experience]. / Povezanost izrazaja biljega CD34 s morfoloskim znacajkama blasta u akutnoj promijelocitnoj leukemiji i klinickim znacajkama oboljelih: iskustvo jednog centra.

The aims of the study were to investigate the association between cytomorphology and immunophenotypic expression of CD34 cell surface antigen of blasts and their relationship with clinical and laboratory characteristics of patients with acute promyelocytic leukemia (APL). Sixteen consecutive patients (male 69% and female 31%) diagnosed with APL at Department of Hematology, Merkur University Hospital between August 1998 and December 2010 were included in the study. The mean age of patients was 43.9 (range: 18-78, SD 14.9). The patients' clinical and laboratory features, cytomorphological characteristics of APL-blasts and their immunophenotype determined by flow cytometry were analyzed. Patients were divided into two groups, CD34- and CD34+, and were then compared according to clinical and laboratory characteristics. There was no difference according to age, sex or white blood cell count between two groups. The mean value of hypogranular/agranular APL-blasts was markedly higher in CD34+ group than CD34- group (34%, range 9-60, SD 24.4 vs. 11.5%, range 0-38, SD 13.7), with borderline statistical significance (P=0.055). CD34- patients had significantly better overall survival than CD34+ ones (P=0.02). Patients without Auer rods detected in APL-blasts had higher CD34 expression (69.4% +/- 33.8) compared to patients with detected Auer rods (7.3% +/- 24.8), but statistical significance was not reached (p=0.053). Our results are consistent with the results of other published studies and point to the fact that higher CD34 expression and lower cytoplasmic granularity of APL-blasts are factors that seem to define a specific subgroup of APL patients. Together with other diagnostic tools currently available, they could be of value in planning treatment of APL patients.

[Isolated myeloid sarcoma involving the mediastinum]. / Izolirani mijeloidni sarkom medijastinuma.

Myeloid sarcoma is a rare extramedullary solid tumor consisting of immature myeloid cells and most commonly involving the bone, skin, lymph nodes, soft tissue, gastrointestinal tract and testis. Mediastinal myeloid sarcoma is very rare. There are two major types of myeloid sarcoma: granulocytic sarcoma and monoblastic sarcoma, according to immature cell type. Myeloid sarcoma is found in 2%-8% of patients with acute myeloid leukemia (AML). Myeloid sarcoma may develop before or concurrently with AML, or may be the initial manifestation of AML relapse in previously treated patients. Blast transformation of some form of myeloproliferative neoplasm or myelodysplastic syndrome may also manifest as myeloid sarcoma. A major differential diagnostic problem is isolated primary myeloid sarcoma without bone marrow and peripheral blood involvement, which may precede leukemic stage for months or years, and which is frequently misdiagnosed, mostly as malignant lymphoma. A case is presented of a 56-year-old female patient complaining of weakness, vertigo, dry cough and breathing difficulties. Clinical examination revealed enhanced vascular pattern on the right chest and right arm edema. Computed tomography (CT) of the thorax showed an expansive growth measuring 11 cm craniocaudally in the anterior mediastinum. Fine needle aspiration cytology of tumor mass yielded a scarcely cellular sample with individual atypical immature cells, fine chromatin structure and scarce cytoplasm with occasional granules and Auer rods. Considering the morphological, cytochemical and immunocytochemical characteristics of immature cells, the diagnosis of myeloid sarcoma was made and verified by histopathology of tumor biopsy sample. Immature cells were not found by analysis of bone marrow puncture sample, immunophenotyping of bone marrow cells and bone biopsy analysis. As immature cell proliferation was not detected in bone marrow and peripheral blood, while spread of the disease beyond the mediastinum was ruled out by imaging methods (CT, ultrasonography), it was decided to be a primary non-leukemic form of mediastinal myeloid sarcoma. Myeloid sarcoma should be taken in consideration on differential diagnosis of solid tumors because making an accurate diagnosis is necessary for timely initiation of appropriate therapy. Weakly expressed or lacking clear signs of myeloid differentiation may hamper morphological diagnosis. As isolated myeloid sarcoma is a very rare entity frequently resembling lymphoma in clinical presentation, it poses a major diagnostic challenge for both morphologists and clinicians.

[Longterm indolent course of pleomorphic mantle cell lymphoma with multiple chromosomal abnormalities]. / Dugogodisnji indolentni tijek pleomorfnog limfoma plastene zone s multiplim kromosomskim abnormalnostima.

Mantle cell lymphoma (MCL) is a B-cell neoplasm characterized by aggressive clinical course with an average 3- to 5-year patient survival. We present a patient whose illness turned from initial classical morphological variant to a more aggressive pleomorphic form of MCL in only a few months, but with unchanged long-term indolent clinical course. At the time when lymphoid cell pleomorphism was proven, the disease presented itself through recurrent peripheral lymphadenopathy without extranodal involvement or general symptoms. Other numerous abnormalities were found next to typical cytogenetic translocation t (11,14). Histopathology confirmed the diagnosis of MCL, pleomorphic type. After autologous stem cell transplantation, the disease remained morphologically the same, but the patient was in a good general condition for a long period of time. More than two years after the pleomorphic MCL had been diagnosed and one year after the transplantation, major lymphadenopathy occurred. Our case report points to a large spectrum of morphological and cytogenetic variability of MCL, which often does not correlate with the clinical course of the disease.

Flow cytometry immunophenotyping (FCI) of fine needle aspirates (FNAs) of lymph nodes.

Flow cytometry immunophenotyping (FCI) has an important role in the clinic work-up of fine needle aspirates (FNAs) of lymph nodes. Its standardization has been defined by proposed analytical protocols and procedures used to assure proper analytical results also in those non-routine samples. In Institute of Clinical Chemistry, "Merkur" University Hospital, FCI is accredited method according to laboratory accreditation standard ISO 15189. According to this laboratory accreditation standard, participation in external quality assessment (EQA) programs is a prerequisite for assuring integrity and quality of the entire laboratory process. A critical analysis of our institutional experience in the feasibility of FCI of the material obtained by FNA of lymph nodes with suspected lymphoma represented the purpose of the study. During an eight-year period in Institute of Clinical Chemistry, "Merkur" University Hospital, a total of 1295 FNA analysis was done, 245 of them with a possible diagnosis of B-cell Non-Hodgkin lymphomas (B-NHL) formed the basis of the study. Lymphocytes were isolated on density gradient according to Boyum et al. The average feasibility of FNAs for FCI analysis was 86% (ranged 78-93%). An acceptable total cell number in FNAs for FCI analysis (4257) was established. In total population of respondents statistical significances in expressions of cellular antigens CD3, CD5, CD22, CD23, CD19 and CD5 on B-cells (CD5+CD19+) between patient's with final diagnosis of benign, reactive lymphoid proliferations and patient's with diagnosis of B-NHL were found. EQA results analysis showed that all results were either inside target values (X +/- 1SD) or inside accepted values (X +/- 2SD). Compatibility of the restriction of immunoglobulins light chains determinated by FCI and cytomorphology diagnosis depends on the choice of criterion values of the light chains ratio which determine the monoclonality. According to the matrix of shares of all classified data of retained neural network, ranges of diagnostic sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and prevalency of 82%, 72%, 93%, 48%, and 72% were produced. As a conclusion, FCI is a reliable methodology for phenotyping FNAs of lymph nodes with suspected B-NHLs detecting their clonality easily.

Primary gastrointestinal non-Hodgkin lymphoma in adults: clinicopathologic and survival characteristics.

Primary non-Hodgkin lymphomas of gastrointestinal tract (PGI-NHL) are the most common extranodal lymphomas with an increasing incidence. The incidence, clinicopathologic characteristics, treatment and survival were assessed in 39 successive, newly diagnosed PGI-NHL patients (23 male and 16 female) treated at "Merkur" University Hospital. The aim of the study was to precisely evaluate their characteristics and compare them with the results reported from other similar studies. The most common site of PGI-NHL was stomach (n = 29, 74%), followed by small intestine (n = 5, 13%), and colon and rectosigmoid (n = 5, 13%). According to the Ann Arbor classification, 34 (87%) patients had stage IE and IIE, and five patients (12%) stage IIIE and IVE. According to World Health Organization (WHO) classification, 29 (87%) patients had diffuse large B-cell lymphoma (DLCBL), two had mantle cell lymphoma, and seven (18%) had marginal zone B-cell lymphoma-mucosa associated tissue (MALT). Twenty-six (66%) patients underwent surgical resection followed by chemotherapy, ten (26%) were treated with chemotherapy alone, and three (8%) were treated surgically. Complete remission was achieved in 28 (72%) and partial remission in seven (18%) patients. Four (10%) patients had progressive disease. In our patients, the major prognostic factor for outcome was the stage of disease. Patients with localized lymphoma (stage IE and IIE) had a significantly longer overall survival: 85% at five years and 65% at ten years. Patients with extended disease (stage IIIE and IVE) had overall survival less than 33%. The prognostic power of erythrocyte sedimentation rate (ESR), total protein, serum albumin, LDH concentration and activity was analyzed. Of these parameters, only LDH had a statistically significant effect on overall survival. In conclusion, our patient group was comparable to other literature reports on PGI-NHL patients according to clinicopathologic characteristics. Disease stage and LDH were the only parameters that had a statistically significant effect patient survival.

Serum immunoglobulins in non-Hodgkin's lymphoma patients.

Serum proteins and immunoglobulin (Ig) findings in 119 non-Hodgkin's lymphoma (NHL) patients were analysed. Out of them 96 (81%) patients had B non-Hodgkin lymphoma (B-NHL), and 23 (19%) T-NHL. Indolent type of NHL was more frequent (77 patients, 65%), then aggressive type of NHL (42 patients, 35%). Most patients had normal serum protein concentration, the increased protein concentration was seen in 17% of patients while decreased concentration was noticed in 7% of patients. Hypoalbuminaemia was more frequent (43%) then hyperalbuminaemia (1%). In contrast to albumin, low levels of other protein fractions (alpha1-, alpha2-, and beta-globulin) were rather rare (0.6%, 4%, and 3% of patients, respectively) and high levels were frequent (23%, 37%, and 8%, respectively). Polyclonal hyperimmunoglobulinaemia was more frequent finding than hypoimmunoglobulinaemia. In 29% patients higher IgG level and in 25% patients higher IgA level were found. IgM hypoimmunoglobulinaemia (22%) was more frequent than IgG (11%) and IgA (8%) hypoimmunoglobulinaemia. M-spike in serum protein electrophoresis was found in 11 (7%) patients. The statistically significant association was not found between serum Ig concentration and lymphoma malignancy grade as well as between serum Ig concentration and immunologic origin of lymphoma. T-NHL patients have more often IgA concentration level above or under normal values than B-NHL patients (p < 0.05).

Myeloid sarcoma involving the breast.

Myeloid sarcoma is a tumor mass with extramedullary growth pattern, composed of myeloblasts or immature myeloid cells. The development of myeloid sarcoma may precede or concur with acute or chronic myeloid leukemia (AML or CML) or other myeloproliferative diseases or myelodysplastic syndromes (MDS). Isolated myeloid sarcoma of the breast is very rare. A case is presented of a 25-year-old, previously healthy woman that presented to our department for a palpable node, 5 x 2 cm in size, in the upper medial quadrant of her left breast. Fine needle aspiration (FNA) produced a sample consisting of medium sized blasts. Additional work-up revealed anemia, thrombocytopenia and leukocytosis, along with atypical blasts detected in peripheral blood and bone marrow smear. Based on the morphology, cytochemical characteristics and immature cell immunophenotype, it was considered a case of acute myeloid leukemia without maturation. In spite of intensive chemotherapy, the patient died within a year of diagnosis. In cases of isolated breast myeloid sarcoma, the diagnosis can be missed if the possibility of myeloid sarcoma is not remembered on differential diagnosis of a breast neoplasm.

T lymphoblastic leukaemia with an unusual Burkitt lymphoma morphology--a case report.

Precursor T-cell acute lymphoblastic leukaemia (T-ALL)/lymphoma (T-LBL) is a neoplasm with cytological features that include blast cells of medium size, high nuclear cytoplasmic ratio and inconspicuous nucleoli, which are usually TdT (Terminal Deoxynucleotidyl Transferase) positive and variably express T-cell markers. We report a case of T-ALL with atypical cytological presentation which showed lymphoblasts with homogenous nuclear pattern, larger amounts of cytoplasm with vacuoles and prominent nucleoli. A 56-year-old male was hospitalized due to high fever and kidney infection. Further examination confirmed anemia, thrombocytopenia, normal level of white blood cells and high level of lactat-dehydrogenase (LDH). Bone marrow aspiration revealed 87% and peripheral blood 41% of lymphoblasts with cytoplasmic vacuoles which suggested Burkitt lymphoma (BL) morphology. Patient's karyotype showed no chromosomal aberations. Identification of immunophenotype discovered cells which were CD2 and CD3 positive and CD20 negative with focal acid phosphatase activity in 67% of blasts. This excluded Burkitt lymphoma and led to diagnosis of T-ALL. The patient was submitted to two cycles of chemotherapy, autologous stem cell transplantation, and intrathecal chemotherapy, but he died after 10 months because of disease complications (lung aspergillosis and pleural effusion). Our case report showed how morphology alone can be misleading and sometimes is not enough in diagnosing ALL. Beside morphologic criteria, setting correct diagnosis depends on identification of immunophenotype by flow cytometry and cytogenetic-molecular abnormalities. Further improvements in the molecular definition of ALL subtypes, development of new and targeted drugs will improve patient's outcome and prognosis.

Serum levels of VEGF and bFGF in hypoxic patients with exacerbated COPD.

Hypoxia frequently complicates the course of chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the two most potent angiogenic factors and may play a role in adaptation to hypoxia. The aims of the study were to assess the serum levels of VEGF and bFGF and to evaluate their mutual relationship in hypoxic patients with exacerbated COPD. The study group consisted of 50 hypoxic (PaO(2) 53 mmHg) patients with exacerbated COPD. Control groups were 30 stable COPD patients with PaO(2) 70 mmHg, and 30 healthy blood donors. The serum concentrations of VEGF and bFGF were measured using commercial enzyme-linked immunoassay kits. Patients with exacerbated COPD had significantly higher serum VEGF levels (1,089.16 +/- 1,128.03 pg/mL) compared to those with stable COPD (197.68 +/- 178.06 pg/mL) (p < 0.0001) and healthy blood donor group (257.69 +/- 170.4 pg/mL) (p < 0.0001). Serum bFGF levels were significantly higher in the exacerbated COPD group (6.15 +/- 2.56 pg/mL) compared to control groups (p = 0.0001). Basic FGF was undetectable in the stable COPD and blood donor groups. Since VEGF and bFGF correlated significantly with the majority of factors investigated in COPD patients, multivariate analysis was performed. According to the step-wise regression analysis, VEGF was best determined by PaO(2), WBC and IL-6. Basic FGF was best determined by PaO(2) and pH. The highly significant, simple correlation between VEGF and bFGF was lost in multivariate analysis. This suggests that their correlation is not independent, but due to factors that remain in the model after step-wise regression. These are essentially linked to the level of hypoxia. Results of our study suggest that VEGF and bFGF production is stimulated in hypoxic patients with exacerbated COPD. Elevated levels of VEGF and bFGF may activate the process of neoangiogenesis, which may lead to increased perfusion and an improvement in tissue oxygenation in this group of patients.

Value of fine-needle aspiration cytology in diagnosis of Hodgkin's lymphoma and anaplastic large cell lymphoma: one centre experience.

The aim of the study was to determine the value and limitations of cytology in diagnosis of Hodgkin's lymphoma (HL) and anaplastic large cell lymphoma (ALCL) as well as differentiation between these two entities. We analysed the FNA cytodiagnoses and histopathological reports, as well as treatment and survival in 89 newly diagnosed consecutive patients with these lymphomas treated in our clinical department. These patients (40 male, 49 female; age range 16-93 years; 44 in clinical stages I-II; 38 with B symptoms) were diagnosed and treated during a period of 64 months (1.1. 2004-1.5.2009). The FNA cytodiagnoses were available in 86 patients and the pathohistological diagnoses were available in 84 patients. Cytology revealed 65 classic HL, 18 ALCL and three patients in which diagnosis was not informative. Among 65 FNA cytodiagnoses of HL, comparison with histopathology was made in 61 cases and the histopathological diagnoses were as follows: 56 (91.8%) HL; three ALCL; one diffuse large B cell lymphoma and one marginal zone B cell lymphoma. In the group of 18 FNA cytodiagnoses of ALCL eight patients (53.3%) had definitive diagnosis of ALCL (either as T-cell or O type), five (33.3%) of HL and in three cases a histopathological diagnosis could not be made. These results confirm the value of FNA in diagnostic procedure in patients with HL and ALCL, especially in HL group of patients. Since we have an almost uniform group of patients according to therapeutic approach, we did univariate analyses and found out that patients with FNA cytodiagnoses of HL, younger than 55 years, with early stage of the disease and without B symptoms had significantly longer overall survival (OS). FNA cytodiagnosis has clinical relevance in differentiation between HL and ALCL.

Post-thaw viability of cryopreserved hematopoietic progenitor cell grafts: does it matter?

Cell viability in peripheral blood progenitor cell (PBPC) grafts and its influence on the clinical course following transplantation was evaluated in 81 consecutive transplantations (72 autologous, 9 allogeneic) performed in patients with hematological diseases. Viability of cells in PBPC grafts immediately upon collection was 98.6 +/- 3.5%, after addition of dimethyl sulfoxide (DMSO) 73.3 +/- 21.8%, and post-thaw 65.2 +/- 16.1%. It did not differ significantly between patients with different diagnoses, gender, age, type of priming used, dose of G-CSF administered or number of CD34+ cells collected. However grafts stored for more than 60 days showed lower post-thaw viability compared to the ones thawed in the 60 days following cryopreservation (56.61 +/- 15.2% vs. 67.6 +/- 15.5%, p = 0.04). Post-thaw graft viability did not influence engraftment time, but there was a predisposition towards infectious complications in the post-transplant period in patients receiving grafts with lower percentage of viable cells. They developed febrile neutropenia more often (72.2% vs. 50% of patients, p = 0.05) and had more febrile days (2.4 +/- 2.6 vs. 1.5 +/- 2.3, p = 0.05) following transplantation. We have demonstrated that PBPC grafts are capable of long term engraftment regardless of the graft storage time or percentage of viable cells post-thaw, which confirms the robustness of CD34+ cells during the freeze/thaw procedures carried out in daily clinical practice. Granulocyte concentration in PBPC grafts could have an influence on infectious complications following transplantation and needs to be further investigated on a larger number of patients.

Diffuse large B-cell lymphoma in patient after treatment of angioimmunoblastic T-cell lymphoma.

Relatively few cases of Epstein-Barr (EBV)-positive B-cell lymphomas arising in patients with angioimmunoblastic T-cell lymphoma (AITL) have been reported. We report a case of AITL in which diffuse large B-cell lymphoma arose 13 months after the initial diagnosis of AITL. In a 36-year-old female patient, evaluated for moderate leukocytosis, peripheral and abdominal lymphadenopathy AITL was diagnosed in March 2008, based on results of fine-needle aspiration cytology (FNAC) of the enlarged cervical and supraclavicular lymph nodes. The diagnosis was also confirmed by immunophenotyping and histopathology of the cervical lymph nodes. The patient initially recieved FED chemotherapy (fludarabine, cyclophosphamide, dexamethasone) followed by elective autologous hematopoietic stem cell transplantation. In April 2009 the patient was hospitalized because of fever, pancytopenia, hyperbilirubinemia and peripheral lymphadenopathy. The FNAC of the enlarged cervical lymph nodes was performed again, but this time the smears were composed of polymorphous population of lymphocytes with the predomination of large cells, CD20+ on immunocytochemical stains. The immunophenotyping confirmed a predomination of monoclonal mature B-cells. Patient had high number of EBV DNA copies in plasma and serologic testing revealed increased titers of EBV VCA IgG and EBV EBNA IgG. CHOP-R chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab) was then administered, resulting in good partial response of the disease. Reduced intensity allogeneic stem cell transplantation performed thereafter, resulted in complete remission of the disease. AITL is a rare lymphoproliferative disorder in which the neoplastic T-cells represent the minority of the lymph node cell population and almost all cases harbor EBV-infected B-cells. Various authors postulated that immunodeficiency in AITL patients together with immunosuppressive effects of cytotoxic drugs, may be responsible for EBV-induced proliferation of latently or newly EBV-infected B-cells with eventual clonal selection and progression to aggressive B-cell lymphoma.

An unusual presentation of Gaucher's disease: aortic valve fibrosis in a patient homozygous for a rare G377S mutation.

Gaucher's disease (GD) has variable presentations, but cardiac involvement is a generally uncommon clinical manifestation of the disease. In the past 25 years, the underlying genetic disorder in GD has been well characterized, with almost 300 mutations identified in the glucocerebrosidase gene (GBA). Nevertheless, clear genotype-phenotype correlations have been confirmed only for the most frequent mutations. We present a female patient, who was known to have aortic valve pathology from the age of 30. Despite medical follow up, at the age of 60 she presented with heart failure (NYHA III). At that time echocardiography showed severe fibrosed aortic valve stenosis. Valvuloplasty was planned, when thrombocytopenia, previously considered to be autoimmune, became severe. Anemia and leukopenia were also noted. Moderate splenomegaly and severe bone marrow infiltration were found on MRI. Bone marrow aspiration revealed typical Gaucher cells and the enzyme activity assay confirmed the diagnosis. DNA investigation showed that the patient is homozygous for the G377S mutation. To our knowledge, of all mutations identified so far, only homozygosity for the D409H mutation has been associated with cardiovascular valvular disease in patients with a rare type 3c GD. G377S, found in our patient, is a rare mutation, previously reported as a 'mild' mutation, because of the finding that homoallelic patients were essentialy asymptomatic or had mild disease. Our patient, also homozygous for G377S mutation, had a severe form of type 1 GD, with rare cardiac valve involvement, which is a previously unreported clinical presentation for this mutation. This case further proves that patients with the same genotypes can have different phenotypes, emphasizing the influence of other genetic and/or environmental factors.