Dariusz Wolowiec

CDK1 and Cyclin A Expression is Linked to Cell Proliferation and Associated with Prognosis in Non-Hodgkin's Lymphomas

Cellular proliferation is regulated by several kinasic complexes associating cyclins and their catalytic subunits cyclin-dependent kinases (CDKs). In order to gain insight into the mechanisms underlying proliferation in non-Hodgkins lymphoma (NHL), we examined the expression of certain cell cycle regulatory proteins normally expressed in lymphoid cells, cyclins A, B, D3 and E and cdk1, 2, 4, and 6. In 70 patients presenting a previously untreated lymphoma, cyclins and CDKs were studied by Western blotting and quantified by densitometry. Flow cytometry study of DNA content was carried out for all patients in order to study cell proliferation and level of ploidy. The results were analysed according to the histological types, the immunological phenotypes of the lymphomas and the outcome of the patients. Cdkl and cyclin A were correlated with the percentage of cells in S and S+G2/M phases, and significantly different according to the grade of malignancy, with the lowest expression in low-, and the highest in high-grade NHL according to the Working Formulation. In B-NHLs, cdk1, cyclin A, as well as cdk2, cyclin D3 and E expression was higher in the aneuploid than in the euploid group. Our results point to some particularities of cell cycle regulation in two lymphoma sub-types: 1) a low expression of cyclin D3 and cdk6 in mantle cell lymphomas and 2) a discrepancy between the high proliferative activity and the level of protein expression in Burkitts lymphomas. CDK1 and cyclin A showed a significant prognostic value for achievement of complete remission (Cdk 1) and for both disease free (cyclin A) and overall survival (cyclin A and cdk1): low protein level was associated with the best prognosis in B-NHLs. Our results show that differential cell cycle regulating protein expression may be associated with different biological and clinical behaviour of NHLs and confirm the usefulness of the study of cell cycle regulation as a tool for understanding lymphoid malignancies.

Increased expression of vascular endothelial growth factor (VEGF) in bone marrow of patients with myeloproliferative disorders (MPD)

Angiogenesis is a multistep process of the development of capillaries from established blood vessels. Angiogenesis probably plays a significant role in the development and progression of hematopoietic malignancies. Higher microvascular density and increased serum levels of proangiogenic factors such as vascular endothelial growth factor (VEGF) or basic fibroblasts growth factor (bFGF) have been reported in acute and chronic leukemias, myeloproliferative and myelodysplastic disorders, multiple myeloma and lymphomas. The microvessel density of bone marrow stroma in myeloproliferative disorders is increased and VEGF is considered as the most potent endothelial cell activator. The purpose of this study was to examine the expression of VEGF in bone marrow of patients with MPD. 60 paraffin-embedded bone marrow core biopsy specimens from newly diagnosed patients with MPD were evaluated. In addition 10 bone marrow core biopsy specimens from adult patients without evidence of malignancy were used as controls. Bone marrow sections were stained immunohistochemically for VEGF (PharMingen, USA). Obtained data show that MPD are associated with an increased expression of VEGF in the bone marrow. This observation support previous studies suggesting that angiogenesis may play a role in the pathophysiology of myeloproliferative disorders. Clinical significance of this phenomenon needs further investigation however thus provides rationale for use of angiogenesis inhibitors in MPD therapy.

A subpopulation of malignant CD34+CD138+B7-H1+ plasma cells is present in multiple myeloma patients

OBJECTIVE It is generally assumed that plasma cells from multiple myeloma (MM) patients do not express the stem cell marker CD34. This assumption has led to several clinical trials based on autologous CD34(+) cell transplantation. However, the results of these trials have been disappointing. MATERIALS AND METHODS We investigated the presence of CD34(+) cell populations in RPMI 8226, KARPAS 417, and U266 MM cell lines in vitro and during their growth as plasmacytoma tumors in nonobese diabetic severe combined immunodeficient mice, and in plasma cells isolated from the bone marrow of 38 MM patients. RESULTS We showed that in both patients and cell lines, a small population of plasma cells expresses CD34. These cells display morphological characteristics of MM plasma cells, are CD19-negative, and express B7-H1 (PD-L1), a T-cell inhibitory molecule. In patients, CD34(+)CD138(+) cells expressed Ki67, a marker for proliferation. Moreover, when cells from the human myeloma cell line U266 were injected into nonobese diabetic severe combined immunodeficient mice, the U266-derived plasmacytoma tumors showed a large CD34(+)CD138(+) Ki67(+) cell population, indicating that these cells were not quiescent in vivo. CONCLUSIONS MM patients carry a small subpopulation of cycling CD34(+)CD138(+)B7-H1(+) plasma cells. Their presence may limit the clinical benefits of autologous CD34(+) cell transplantation.

Circulating sCD138 and some angiogenesis-involved cytokines help to anticipate the disease progression of early-stage B-cell chronic lymphocytic leukemia.

Syndecan-1 (CD138) is a transmembrane heparin sulfate proteoglycan expressed on distinct stages of differentiation of B-lymphoid cells. Its prognostic value in B-cell chronic lymphocytic leukemia (B-CLL) has not been evaluated so far. The serum concentration of sCD138 and some angiogenesis-involved cytokines: vascular endothelial growth factor (VEGF), basis fibroblast growth factor (bFGF), and endostatin were studied in 52 previously untreated patients with B-CLL. We found that bFGF and sCD138 levels were significantly higher in B-CLL patients than in controls. In patients with sCD138 level or endostatin level below the median value the lymphocyte count was higher than in patients with serum level of those cytokines above the median value. In patients with progressive disease bFGF level was significantly higher and sCD138 level significantly lower than in patients with stable one. Moreover, high sCD138 level was associated with longer lymphocyte doubling-free survival, and, on the limit of statistical significance, a high endostatin level was associated with shorter progression-free survival. We conclude that serum sCD138 level is increased in early stage B-CLL patients and may have a positive prognostic value as to the dynamics of the disease.

Plasma cell proliferation in monoclonal gammopathy: Relations with other biologic variables—Diagnostic and prognostic significance

PURPOSE We investigated the place of direct plasma cell proliferation analysis beside other biologic data in monoclonal gammopathy, particularly the serum level of C-reactive protein (C-RP) PATIENTS: Eighty patients were studied at the time of their diagnosis. Patients with a serum creatinine level greater than 200 mumol/L were excluded. METHODS Plasma cell proliferation analysis was performed after bromodeoxyuridine incorporation and double immunoenzymatic labeling on cytological smears, making determination of the plasma cell labeling index (LI) possible. The other biologic variables studied were related to tumor burden (plasmacytosis, hemoglobin, serum levels of monoclonal immunoglobulin, albumin) or to kidney function (creatinine). beta 2-microglobulin (beta 2-M), C-RP, lactic dehydrogenase (LDH), and calcemia were also assessed. RESULTS No correlation was found between LI and serum C-RP. LDH was the sole variable significantly correlated to C-RP (P < 0.01). Besides the biologic parameters used for the staging according to the Durie and Salmon classification, beta 2-M, albumin and LI were significantly different between stages (P < 0.0002, < 0.0004, < 0.00001). LDH and C-RP showed no significant difference. Results were similar when patients with and without bone lesions were analyzed separately. Multivariate analysis ranked these variables as follows with respect to prognostic value: beta 2-M, LI, and age. CONCLUSION Among the variables analyzed, LI is the sole true reflector of cell proliferation. We confirm its diagnostic and prognostic value.

CD8+CD28− Lymphocytes in Peripheral Blood and Serum Concentrations of Soluble Interleukin 6 Receptor are Increased in Patients with Graves’ Orbitopathy and Correlate with Disease Activity

Background. The extrathyroid, orbital manifestation of Graves’ disease (GD)—Graves’ orbitopathy (GO)—presents a difficult clinical problem. The immunological status of GO patients is still under investigation. The aim of this study was to assess the serum concentration of interleukin 6 (IL-6), soluble interleukin 6 receptor (sIL-6R), and CD8+CD28− lymphocytes in GO patients and to evaluate if these parameters were associated with disease activity. Patients. Thirty-nine patients (29 women and 10 men, aged 24–71, mean 50.18) with newly diagnosed GD were enrolled in the study. Active GO was diagnosed in 20 patients. The control group included 12 healthy individuals. Methods. Serum concentrations of IL-6 and sIL-6R were estimated by ELISA. Percentages of CD8+CD28− lymphocytes in peripheral blood were assessed by flow cytometry. Results. Mean serum IL-6 and sIL-6R concentrations were significantly higher in all GD patients and in GO and non-GO patients than in normal controls. In all GD patients and the non-GO group, serum IL-6 and sIL-6R concentrations were significantly reduced after efficient treatment. In GO patients, only serum sIL-6R concentration was significantly lower after efficient treatment. In all GD patients, the mean percentage of CD8+CD28− lymphocytes was significantly lower after efficient treatment. In GO patients, the mean percentage of CD8+CD28− lymphocytes was significantly higher than in the non-GO group or in normals. Moreover, in the GO group, the mean percentage of CD8+CD28− lymphocytes was significantly lower after treatment. Conclusion. Our results have shown that CD8+CD28− lymphocyte percentage in peripheral blood and serum concentration of sIL-6R are increased in GO patients and correlate with disease activity.

High intracellular content of cyclin‐dependent kinase inhibitor p27Kip1 in early‐ and intermediate stage B‐cell chronic lymphocytic leukemia lymphocytes predicts rapid progression of the disease

Previous studies showed that peripheral blood lymphocytes of B‐cell chronic lymphocytic leukemia (B‐CLL) displayed a high intracellular level of cell cycle inhibitory protein p27Kip1. It has been suggested that its’ high expression may confer them survival advantage and lead to unfavorable prognosis, but the prognostic significance of p27Kip1 expression for previously untreated, non‐advanced stage B‐CLL was not established. We studied a relationship between the intracellular level of p27Kip1 of lymphocytes of early‐ and intermediate stage B‐CLL patients and their spontaneous apoptosis in vitro, as well as prognostic significance of p27Kip1 in B‐CLL lymphocytes for the risk of disease progression. Intracellular p27Kip1 content of peripheral blood lymphocytes obtained from 48 previously untreated 0–II Rai stage B‐CLL patients was determined by flow cytometry. The viability and apoptosis of those lymphocytes after 72‐h culture were also assessed. During the follow‐up period (6–71 months, median 59.5), we recorded the time elapsed to the doubling of lymphocyte count, progression to a higher Rai stage and the appearance of indications for cytostatic treatment. The p27Kip1 expression was neither correlated with initial lymphocyte count, CD38 expression, cell viability nor spontaneous apoptosis ratio after 72‐h culture. Higher p27Kip1 level was related to the probability of earlier occurrence of each of three above‐mentioned events. We did not find a prognostic significance of in vitro cell viability nor apoptosis as to the risk of disease progression. Our results indicate that elevated intracellular p27Kip1 level in leukemic lymphocytes of early‐ and intermediate stage B‐CLL patients contributes to rapid progression of the disease.

Re-treatment with cladribine-based regimens in relapsed patients with B-cell chronic lymphocytic leukemia: Efficacy and toxicity in comparison with previous treatment

Abstract: The aim of the study was to determine the effectiveness and toxicity of cladribine (2‐CdA) used alone or in combination with prednisone (P) or cyclophosphamide and mitoxantrone in re‐treatment of patients with progressive B‐cell chronic lymphocytic leukemia (B‐CLL). We analyzed treatment outcome in 40 patients who had responded to previous treatment with 2‐CdA‐based regimens. Criteria for re‐treatment were the same as for the first treatment. The patients were retreated with the same agents if they responded to the first treatment and then relapsed with progressive disease not earlier than 3 months after achieving the first response. Eight patients received 2‐CdA alone (0.12 mg kg−1 d−1) i.v. for 5 d, and 21 patients additionally were given P (30 mg m−2 d−1) orally, also for 5 d. Eleven patients received 2‐CdA for 3 d combined with cyclophosphamide (650 mg m−2) i.v. and mitoxantrone (10 mg m−2) i.v. on day 1 (CMC regimen). The cycles were repeated usually at 4 wk intervals or longer if severe myelosuppression or infections occurred. The therapy was finished if complete remission (CR) was achieved or until maximum of six courses. Overall response (OR) in re‐treatment was obtained in 16 out of 40 (40%) patients (95% CI 16–64), including 62% after 2‐CdA, 33% after 2‐CdA +P and 36% after CMC. CR was obtained in four (10%) patients. Residual disease evaluated in the patients with CR by surface immunophenotyping had been demonstrated in 5 out of 16 (31%) patients after the first treatment and in one out of four (25%) patients after re‐treatment. The median progression‐free survival (PFS) was 16 months (range 3–39) for the first treatment and 9.5 months (range 3–18) for re‐treatment (P=0.34). Grade III or IV neutropenia was observed in 20% patients during the first treatment and in 35% patients during re‐treatment (P=0.1). 2‐CdA‐induced thrombocytopenia occurred in 20% and 42% of the patients, respectively (P=0.05). Anemia was also more frequent during re‐treatment (35%) than during the first treatment (7%) (P=0.007). Autoimmune hemolytic anemia developed in four (10%) of the patients during or after re‐treatment. Severe infections, including pneumonia and herpes reactivation, occurred in 11 patients during the first treatment and in 10 patients during re‐treatment. Twelve (30%) patients died during the study. Infections were the cause of death in six and AIHA in two patients. In conclusion, 2‐CdA applied in monotherapy or in combination with prednisone or cyclophosphamide and mitoxantrone has therapeutic activity in some B‐CLL patients in whom these drugs induced earlier complete or partial remission. However, since the second response is usually shorter and myelotoxicity more pronounced than during the first therapy, more clinical trials to find other therapeutical approaches are necessary.

Expression of PIM-2 and NF-κB genes is increased in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and is associated with complete remission rate and overall survival

INTRODUCTION PIM-2 is a proto-oncogene that encodes for a serine/threonine kinase that interacts with various signaling molecules. PIM-2 is highly expressed in neoplastic tissues and in leukemic and lymphoma cell lines, which is consistent with its role during oncogenic transformation. The nuclear factor kappa B (NF-κB) pathway appears to be deregulated in a variety of tumors, with sustained activity of NF-κB leading to apoptotic resistance in tumor cells. The aim of this study was to investigate whether expression of PIM-2 and NF-κB is altered in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS One hundred forty-three patients were included: 91 with AML and 52 with ALL, aged 18-84 (median 46.7). Eighty-three patients (51 AML and 32 ALL) reached complete remission (CR). Bone marrow samples were collected at the time of diagnosis. Control samples were obtained from 24 healthy donors. We analyzed PIM-2 and NF-κB expression by RQ-PCR analysis. RESULTS Expression of both PIM-2 and NF-κB in all leukemia patients and subgroups was significantly higher than in controls. AML patients who reached CR expressed PIM-2 and NF-κB at significantly lower levels than did patients with primary resistance to chemotherapy and who did not reach CR (NCR). Survival analysis revealed that in AML patients with higher expression of PIM-2 the overall survival (OS) was significantly shorter than in patients with lower expression. CONCLUSION Our data indicate that PIM-2 and NF-κB gene expression is increased in patients with AML and ALL. Moreover, high PIM-2 expression is associated with CR rate and OS in AML patients.

Ofatumumab for treating chronic lymphocytic leukemia: a safety profile

Introduction: Ofatumumab, the first fully human IgG1κ, belongs to the second generation of the first class of anti-CD20 monoclonal antibodies. The drug used alone and in combination with drugs having different mechanisms of action has shown a favorable toxicity profile and significant benefit especially in relapsed/refractory chronic lymphocytic leukemia (CLL) patients in doses up to 2000 mg. Areas covered: This article reviews pharmacokinetic, clinical application for CLL treatment, and safety profile of ofatumumab as well as differences and similarity between ofatumumab and rituximab. Publications in English from 2010 through October 2015 were surveyed on the MEDLINE database for articles. Proceedings of the American Society of Hematology held during the last 5 years were also included. Expert opinion: Ofatumumab more effectively than rituximab enhanced complement-dependent cytotoxicity playing the crucial role for its therapeutic activity. The drug is highly effective in the first-line and salvage treatment of CLL, essentially as a part of immunochemotherapy, and probably also as maintenance therapy. Its safety profile is very advantageous, since adverse events are usually limited to grade 1 and 2 infusion-related reactions, which tend to decrease throughout the treatment. Its advantage over the other anti-CD20 monoclonal antibodies in the treatment of CLL remains to be determined in the direct head-to-head trials.