Mustafa Nuri Yenerel

Analysis of p53 Tumor Suppressor Pathway Genes in Chronic Lymphocytic Leukemia

The p53 tumor suppressor gene plays an important role in preventing tumor development. The p53 protein interacts with other p53 signal pathway members to control cell proliferation. In this study, expression of the p53, Human homolog of murine Double Minute 2 (HDM2), p14Alternating Reading Frame (ARF), Zinc Finger and BTB domain containing 7A (ZBTB7A), and B-Cell Lymphoma 6 (BCL6) genes was quantitatively investigated by real-time polymerase chain reaction (PCR) in the peripheral blood of patients with chronic lymphocytic leukemia (CLL) and healthy controls. Plasma fibronectin levels were determined by enzyme-linked immunosorbent assay. Expression of the p53, p14, and HDM2 genes were significantly higher in the patients. However, ZBTB7A and BCL6 gene expression was not detectable in both groups. A positive correlation between p14ARF and HDM2 expression and a negative correlation between p53 and p14ARF expression was observed. Expression of the p14ARF and HDM2 genes were inversely correlated in the control group. Neither HDM2 nor p14ARF gene expression was correlated with p53 expression. The p53 gene was also analyzed for the presence of mutations. A splice-site mutation was found in a single patient. Our findings indicate that expression of the p53, p14ARF, and HDM2 genes are associated with CLL. Elucidation of the mutual interactions at the protein level warrants further studies.

A case of chronic lymphocytic leukemia with massive ascites.

An 81-year old woman with a history of chronic lymphocytic leukemia (CLL) was admitted with night sweats and abdominal distension. A complete blood count showed hemoglobin 5 g/dL, white blood cell (WBC) count 28.5×109/L and platelets 38.4×109/L. Peripheral blood smear examination showed a large number of smudge cells and lymphocytosis composed of mature-looking lymphocytes with clumped nuclear chromatin. Computed tomography scan demonstrated enlarged cervical, axillary, paraaortic, retroperitoneal and mesenteric lymph nodes with concomitant omental thickening and ascites. Also, the liver and the spleen were enlarged in the presence of multiple ill-defined hypoechoic areas in the latter. Histopathological analysis of the cervical lymph node biopsy was consistent with CLL. Bone marrow examination showed diffuse infiltration of the marrow with small lymphocytes. Analysis of the ascitic fluid revealed an exudate with WBC 1220 cells/mL. Cytocentrifuge preparation of the ascitic fluid showed small mature lymphoid cells containing hyperchromatic nuclei with coarsely granular chromatin. On flow cytometric analysis of the ascitic fluid, expression of CD5, CD19, CD20, CD22, CD23, CD45 and HLA-DR was compatible with a diagnosis of CLL, in accordance with the results of the peripheral blood analysis. The patient was treated with chemotherapy consisting of cyclophosphamide, vincristine and prednisolone but died within one month after development of non-chylous ascites.

Massive Ascites as the Initial Manifestation of Mantle Cell Lymphoma: A Challenge for the Gastroenterologist

Involvement of the serosa may be the presenting feature in a wide and complex variety of lymphoproliferative diseases, with differing clinical outcomes covering a spectrum of benign and malignant conditions. Effusions involving peritoneal and pericardial cavities are uncommon during the course of hematological malignancies. Obstructive and/or infiltrative tumor mass or vascular leakage due to stimulation by vascular endothelial growth factor contribute to the pathogenesis. In addition to clinical findings, cytomorphology and flow cytometric immunophenotyping of the serosal fluid yield valuable information in the differential diagnosis of lymphocytic infiltrates. Herein, we describe the case of primary mantle cell lymphoma in a 75-year-old man presenting with abdominal fullness and weight loss, suggesting a gastrointestinal pathology.

Clinical course and disease burden in patients with paroxysmal nocturnal hemoglobinuria by hemolytic status

Disease characteristics of patients enrolled in the International PNH Registry were assessed during two follow-up periods based on hemolytic status while untreated with eculizumab: Non-hemolytic cohort: follow-up time defined as time from disease start until last reported untreated lactate dehydrogenase (LDH) value <1.5×upper limit normal (ULN); Hemolytic cohort: follow-up time defined as time from LDH ≥1.5×ULN at or post-disease start, to most recent untreated follow-up. A total of 1012 patients met criteria for the Non-hemolytic cohort and 1565 patients for the Hemolytic cohort; median (min, max) years of follow-up were 2.2 (0.0, 54.2) and 1.2 (0.0, 37.2) years, respectively. Annual rate of thrombotic events (TEs) was lower in the Non-hemolytic than Hemolytic cohort (0.01 events/person-year vs. 0.03 events/person-year; p<0.001). Mortality was lower in the Non-hemolytic cohort than the Hemolytic cohort (0.1% (1 death) vs. 1.8% (22 deaths); p<0.001). While elevated risks for TEs were observed in patients with hemolysis, many TEs were also observed in patients without hemolysis. As thrombosis is the leading cause of mortality in patients with PNH, this real-world analysis highlights the importance of awareness and monitoring for TEs in patients with PNH regardless of hemolytic status.

Leukemic ascites as an initial presentation of acute myelomonocytic leukemia with inversion of chromosome 16.

To the Editor: The inversion of chromosome 16, inv(16), a cytogenetic abnormality expressed in core binding factor acute myeloid leukemias (AML), is associated with myelomonocytic differentiation and eosinophilia.1 Even though inv(16) generally portends a good prognosis, accompanying mutations detected by molecular genetic methods, such as KIT and Ras mutations, alter their response to treatment.2 Infiltration of leukemic cells into serous effusions is unusual. To our knowledge, there are only a few reports of AML with inv(16) presenting with leukemic ascites.2 We present a 33-year-old woman with jaundice and massive ascites. The laboratory tests showed the following: hemoglobin 8.3 g/dL, hematocrit 25%, total leukocyte count 135 800/mm3, and platelet count 32 000/mm3, erythrocyte sedimentation rate 45 mm/hr, AST 597 U/L, ALT 111 U/L, ALP 417 U/L, GGT 191 U/L, lactate dehydrogenase 6515 U/L, total bilirubin 9 mg/dL and direct bilirubin 8.2 mg/dL. On peripheral blood smear, myeloblasts comprised 67% of the cells and the bone marrow analysis showed 57% myeloblasts with eosinophilic differentiation. Immunophenotypic analysis of the bone marrow was positive for CD13, CD14, CD45, CD33, CD34 and HLA-DR. FISH analysis of the bone marrow revealed an inv(16) signal. The final diagnosis was acute myelomonocytic leukemia (FAB Classification M4e) with inv(16). Abdominal computed tomography revealed massive ascites and multiple lympadenopathies with a maximal diameter of 1.5 cm at the mesenteric region. A diagnostic and therapeutic paracentesis was performed. Analysis of the ascitic fluid showed an exudate with a white blood cell count 3140 cells/ mL; red blood cell count 70000 cells/mL; monocyte count 1910 cells/mL. The ascitic total protein was 3.9 g/dL (serum, 7.1 g/dL), glucose 208 mg/dL (serum, 216 mg/ dL), lactate dehydrogenase 1918 U/L (serum, 2813 U/L) and albumin 2.4 g/dL (serum, 3.9 g/dL). Cytocentrifuge preparation of the patient’s ascitic fluid showed myeloblasts and monoblasts with irregular nuclei and prominent nucleoli (Figure 1). Flow cytometric analysis of the ascitic fluid showed the expression of CD13, CD14, CD33, CD34, CD45 and HLA-DR compatible with the diagnosis of acute

Correspondence: The Influence of Flow Cytometric Gating Strategy and Cell Preparation Procedures on CD34+ Cell Quantification

11 BECAU SE THE SM ALL PO PU LATION of cells that express the CD34 antigen is known to correlate with multilineage engraftment, CD34 1 cell quantification by flow cytometric analysis is of great importance in determining the adequate number of hematopoietic stem cells (HSC) for both autologous and allogeneic peripheral stem cell transplantation (1). Flow cytometric enumeration of CD34 1 stem cells varies according to the differences in the method of cell preparation, the reagents used, and the flow cytometric analysis (2). The proposed Milano and ISHAGE guidelines define different gating strategies for CD34 1 cell analysis by flow cytometry (3,4). We analyzed 11 bone marrow (BM) and 11 peripheral blood (PB) samples obtained from healthy donors and 11 leukapheresed stem cell (LSC) harvest samples to compare the influence of cell preparation procedures on the gating strategies. Briefly, 100 m l of sample (LSC and BM diluted to 10 3 103 cells/ m l) was incubated with 10 m l of anti-CD34-PE (BIRMA-K3, Dako, Carpenteria, CA) and 10 m l of anti-CD45-FITC (T29-33, Dako), followed by isotypic controls for 30 min at 4°C in the dark. Red cells were lysed with lysing solution (FACSLyse, Becton Dickinson, Mountain View, CA), and specimens were analyzed without washing or after one wash with PBS or one wash with PBS 1 2% human albumin or two washes with PBS. Following the ISHAGE guidelines and the Milano protocol, listmode data of each sample were analyzed by two different gating strategies. In the Milano protocol, forward scatter (FSC) and side scatter (SSC) were used to gate the nucleated cells and to exclude the RBC, debris, and cell aggregates (live gate) (3). The gated nucleated cell population was plotted on CD34 versus SSC. Only CD34 1 events with low SSC were used to calculate the number of CD34 1 cells, expressed as a percentage of the gated nucleated cells. In the second gating strategy, proposed by ISHAGE, the first gate selected the CD45 1 events, and a second gate chose the CD34 1 events. CD34 1 cells were selected by their CD34 expression, their characteristic low to in-

Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis

Background: Multiple myeloma is a plasma cell dyscrasia characterized by transformation of B cells into malignant cells. Although there are data regarding the molecular pathology of multiple myeloma, the molecular mechanisms of the disease have not been fully elucidated. Aims: To investigate the gene expression profiles in bone marrow myeloma cells via RNA-sequencing technology. Study Design: Cell study. Methods: Myeloma cells from four patients with untreated multiple myeloma and B cells from the bone marrow of four healthy donors were sorted using a FACSAria II flow cytometer. The patient pool of myeloma cells and the control pool of B cells were the two comparative groups. A transcriptome analysis was performed and the results were analyzed using bioinformatics tools. Results: In total, 18.806 transcripts (94.4%) were detected in the pooled multiple myeloma patient cells. A total of 992 regions were detected as new exon candidates or alternative splicing regions. In addition, 490 mutations (deletions or insertions), 1.397 single nucleotide variations, 415 fusion transcripts, 132 frameshift mutations, and 983 fusions, which were reported before in the National Center for Biotechnology Information, were detected with unknown functions in patients. A total of 35.268 transcripts were obtained (71%) (25.355 transcripts were defined previously) in the control pool. In this preliminary study, the first 50 genes were analyzed with the MSigDB, Enrichr, and Panther gene set enrichment analysis programs. The molecular functions, cellular components, pathways, and biological processes of the genes were obtained and statistical values were determined using bioinformatics tools and are presented as a supplemental file. Conclusion: EEF1G, ITM2C, FTL, CLPTM1L, and CYBA are identified as possible candidate genes associated with myelomagenesis.

Comparison of the cytogenetic and molecular analyses in the assessment of imatinib response in chronic myelocytic leukemia.

We aimed to compare the cytogenetic and molecular analyses in the assessment of imatinib mesylate response in patients suffering the chronic phase of chronic myelocytic leukemia who were refractory to alpha-interferon treatment. A total of 117 patients in the chronic phase of chronic myelocytic leukemia were included. The patients were treated with 400 mg/day imatinib mesylate. Bone marrow samples were obtained for the cytogenetic and molecular analyses. Patients without the Ph chromosome were defined as complete cytogenetic responders. Partial cytogenetic response was determined when the Ph chromosome was detected in 1-35% of the cells. Molecular response was determined by quantitative real-time reverse transcriptase polymerase chain reaction (QR-PCR) and defined as no detection of BCR-ABL mRNA. The frequencies of complete and partial cytogenetic response were 29% (n = 34) and 15% (n = 18), respectively. No cytogenetic response was achieved in 56% (n = 65) of the patients. Molecular response was achieved in 62% (n = 21) and 33% (n = 6) of the complete and partial cytogenetic responders, respectively. All of the 65 patients with no cytogenetic response were also molecular nonresponders. We conclude that there is reasonable agreement between the cytogenetic and molecular analyses. Both methods are complementary in the assessment of response to therapy.

2nd International Congress on Leukemia, Lymphoma and Myeloma.

The 2nd International Congress on Leukemia, Lymphoma and Myeloma (ICLLM) is a novel initiative of the Turkish Society of Hematology. The first congress was organized in Fethiye in May, 2007. After the success and positive impressions of the participants, the THD decided to organize the ICLLM every other year. The major focus was hematological malignancies, not only LLM, but also myeloproliferative disorders and myelodysplastic syndromes. The target audience was hematologists, oncologists and those involved in stem cell transplantation. The congress was held over 2 and a half days and attended by more than 350 participants from 25 different countries. The scientific program was shaped by 12 distinguished international speakers, who cochaired and designed the sessions. The majority of the hematologists are caring for malignant patients and the Congress was a great opportunity for participants to get the latest update before the American Society of Clinical Oncology and American Society of Hematology 2009 official meeting. The rising interest and encouraging increase in the number of participants geared us to announce the organization of 3rd ICLLM in May 2011 in Istanbul.