Standardisation of minimal residual disease in multiple myeloma.

The assessment of the effectiveness of chemotherapy in oncology cannot disregard the concept of minimal residual disease (MRD). In fact, the efforts of numerous scientific groups all over the world are currently focusing on this issue, with the sole purpose of defining sensitive, effective assessment criteria that are, above all, able to give acceptable, easily repeatable results worldwide. Regarding this issue, especially with the advent of new drugs, multiple myeloma is one of the haematologic malignancies for which a consensus has not yet been reached. In this review, we analyse various techniques that have been used to improve the sensitivity of response, aimed at reducing the cut-off values previously allowed, as well as serological values like serum-free light chain, or immunophenotypic tools on bone marrow or peripheral blood, like multi-parameter flow cytometry, or molecular ones such as allele-specific oligonucleotide (ASO)-qPCR and next-generation/high-throughput sequencing technologies (NGS). Moreover, our discussion makes a brief reference to promising techniques, such as mass spectrometry for identifying Ig light chain (LC) in peripheral blood, and the assessment of gene expression profile not only in defining prognostic risk at the diagnosis but also as a tool for evaluation of response.

MDR-1 polymorphisms (G2677T and C3435T) in B-chronic lymphocytic leukemia: an impact on susceptibility and prognosis.

Patients with B-chronic lymphocytic leukemia present diverse clinical features, genetic abnormalities, variable response to treatment, and heterogeneous prognosis. Novel biological markers such as IgVH mutation, CD38, and ZAP-70 expression have shown to offer important prognostic information. An altered expression of the multidrug resistance 1 may represent an additional prognostic marker. Aim of our study was to evaluate two MDR-1 gene polymorphisms: G2677T polymorphism in exon 21 and C3435T polymorphism in exon 26, to evidence if polymorphisms influence the risk of development of B-CLL and whether genomic polymorphisms provide prognostic information on the clinical progression of the disease. A total of 125 patients with B-CLL and 125 healthy subjects were enrolled in this study. The mutant homozygous 2677 TT genotype was found to be associated with the occurrence of B-CLL and higher T allele frequency in patients with B-CLL when compared with controls was observed (P=0.009). When comparing the prognostic patients' characteristics, patients with 2677 GT genotype were statistically linked to the unmutated IgVH genes (r=0.209, P=0.01). Moreover, the same genotype was correlated with lymphocyte number (r=0.269, P=0.02). Finally for the 2677GT polymorphism, the heterozygous status was associated with higher hemoglobin levels (r=0.247, P=0.005). As far the C3435T MDR1 polymorphism, we were not able to identify any significant correlation with IgVH gene status or other variables. In conclusion, MDR1 gene polymorphism could be a factor predisposing to LLC. Moreover, our findings support the possibility of considering these genomic polymorphisms as prognostic markers in patients with B-CLL.

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.

Evaluation of circulating endothelial cells, VEGF and VEGFR2 serum levels in patients with chronic myeloproliferative diseases.

Authors evaluated some markers of angiogenetic activity in patients with chronic myeloproliferative diseases (CMDs). In this study by using a cytofluorimetric analysis we evaluated circulating endothelial progenitor cells (EPCs) in patients with chronic myeloproliferative disease. Moreover, in the same group of subjects, we evaluated serum levels of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR2). In our patients, we have found an increase in the number of endothelial progenitor cells in primary myelofibrosis (PMF) and polycythaemia vera (PV) patients, while an increase of circulating endothelial cells (CECs) was found in all patients with CMD. Moreover, we found higher serum levels of VEGF with respect to control subjects in every group of patients with CMD, and a not significant reduction of VEGFR2 levels in essential thrombocythaemia (ET) patients. A correlation was also found in PV patients between VEGF levels and erythrocyte number and in PMF subjects with the count of white cells. Our data suggest that some markers of angiogenesis are activated in CMD patients and angiogenesis may have a role in the pathophysiology of chronic myeloproliferative disorders.

Patients with bisphosphonates-associated osteonecrosis of the jaw have reduced circulating endothelial cells.

Osteonecrosis of the jaws (ONJ) associated with the use of bisphosphonates is a newly described entity. To elucidate the mechanism leading to ONJ and to test the hypothesis that in patients with ONJ the bisphosphonates may interfere with endothelial cell proliferation, using flow cytometric analysis we evaluated the number of circulating endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) in eight patients with bisphosphonate treatment and osteonecrosis, eight multiple myeloma (MM) patients with bisphosphonates treatment without ONJ and five normal subjects. MM patients showed an increase of CD34+ cells with respect the control subjects and ONJ subjects. EPCs and CECs were higher in MM patients compared to controls and ONJ patients. ONJ patients showed a decrease of EPCs compared to control subjects while CECs were similar to the controls group. Our results seem to show the possibility that bisphosphonates could have a antiangiogenic effect and a suppressive effect on CECs of patients with ONJ.

Bisphosphonate-associated osteonecrosis of the jaw in patients with multiple myeloma and breast cancer.

Osteonecrosis of the jaw is an unremitting adverse outcome associated with bisphosphonate therapy in patients with multiple myeloma or bone metastases from solid tumors. Twelve patients who presented with exposed bone associated with bisphosphonates were reviewed to determine the type, dosage and duration of their bisphosphonate therapy, presenting findings, comorbidities and the event that incited the bone exposure. The discontinuation of bisphosphonate therapy has not helped reverse the presence of osteonecrosis, and the surgical manipulation of the involved site appears to worsen the underlying bone pathology. Hyperbaric oxygen, which has proven efficacious in other forms of osteonecrosis by establishing an oxygen gradient, is of no definitive benefit to patients with bisphosphonate-induced exposed bone. Antibiotic therapy is useful in controlling pain and swelling but ineffective in preventing the progression of the exposed bone. To date, prevention is the only currently possible therapeutic approach to the management of this complication.

Differential levels of soluble angiopoietin-2 and Tie-2 in patients with haematological malignancies.

The system involving angiopoietin-2 (Ang-2) and its receptor, Tie-2, appears to play an important role not only in tumor angiogenesis, but also in the biology of haematological and non-haematological malignancies. In the present study we evaluated the serum levels of soluble Ang-2 (sAng-2) and soluble Tie-2 (sTie-2) in patients with haematological malignancies. Measurements were carried out in 15 patients with chronic myeloid leukaemia (CML), 25 with essential thrombocythemia (ET), 24 with multiple myeloma (MM) and six with monoclonal gammopathy of undetermined significance (MGUS). In addition, we correlated the levels of angiopoietins with known prognostic factors. sAng-2 and sTie-2 were quantified with enzyme-linked immunosorbent assay (ELISA). In patients with CML and MM the levels of sAng-2 were significantly higher (1686.53 +/- 936.41 pg/mL and 1917.82 +/- 1427 pg/mL, respectively) than in controls (n = 15; 996.096 +/- 414.65 pg/mL) (P < 0.01). In patients with MM sAng-2 levels were significantly increased with increasing stage of disease, from stage I to stage III (P < 0.03) and presented a trend of correlation with Beta2-microglobulin levels (r = 0.317) and grade of bone involvement. Furthermore, the levels of sAng-2 determined after 6 months of chemotherapy in CML patients were significantly lower than at diagnosis in the patients who achieved haematological remission. Circulating sTie-2 levels were increased in patients with ET (17.5 +/- 9.2 vs 9 +/- 3.5 ng/mL; P < 0.01) and in those with CML (16.29 +/- 8.7 ng/mL; P < 0.04). In conclusion, abnormal levels of sAng-2 and sTie-2 are present in some haematological malignancies. These markers may play a role in the pathophysiology of these conditions and their progression.

Soluble angiogenic factors: implications for chronic myeloproliferative disorders.

The role of angiogenesis for the progressive growth and metastatic process of tumours is well established. What is not clear, though, is the clinical prognostic significance of the angiogenic factors in malignant haematological diseases. In this study, we have assessed the plasma and serum levels of two major angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) in 55 patients affected by chronic myeloproliferative disorders (CMD). This series included 25 patients with essential thrombocythemia (ET), 10 patients with chronic myelocytic leukaemia (CML), 14 patients with polycythemia vera (PV), and 6 patients with primary myelofibrosis (MF), and they were compared to 20 healthy control subjects. In all patients the plasma VEGF concentration was significantly increased to the healthy control group (P < 0.004). The highest concentrations were found in the patients with ET (178.25 +/- 125.22 pg/ml). The VEGF levels were significantly higher in CMD patients with vascular complications than those in CMD patients without complications (P < 0.01). The b-FGF serum levels also appeared to be significantly higher in almost all the CMD patients compared to the control group (P < 0.07). A significant correlation was found between the VEGF levels and the platelet count in the ET patients and the spleen index in the CML patients. VEGF level, in this study, is associated with increased risk of thrombotic complications. There is evidence of increased levels of soluble angiogenic factors in malignant haematological disorders, but their contribution to the progression of diseases is yet unclear.

Myeloproliferative Disease: Markers of Endothelial and Platelet Status in Patients with Essential Thrombocythemia and Polycythemia Vera.

Vascular complications are the main cause of morbidity in polycythemia vera (PV) and essential thrombocythemia (ET). To investigate plasma concentrations of soluble P-selectin (sP-Sel.), soluble E-selectin (sE-Sel.) and soluble thrombomodulin (sTM) in relation to the presence of thromboembolic events 38 patients with Chronic Myeloproliferative Disorders (CMD) (14 PV pts and 24 ET pts), 15 age - matched controls and 15 patients with secondary thrombocytosis were studied. Plasma levels of P-Sel., E-Sel. and TM were significantly increased in the group of patients as compared with control subjects (respectively p < 0.001, p < 0.04 and p < 0.01). sP-Sel. levels showed no significant difference between the patients and those with secondary thrombocytosis. No difference in sP-sel levels were also observed between subgroups of CMD patients with and without vascular complications. However, among patients with ET, those with thrombosis had higher sP-Sel levels than those without thrombosis (1.177 +/- 110.48 ng/ml vs 816.25 +/- 99.27 ng/ml). High levels of sE-Sel and sTM were found in CMD patients (71.93 +/- 39.08 ng/ml and 35.81 +/- 20.79 ng/ml, respectively). Plasma sE-Sel. concentration was significantly higher in CMD patients with thrombosis than that in CMD patients without thrombosis (p < 0.001). There was no difference in sTM concentration between two groups. These findings indicate that sustained endothelium and platelet activation is present in patients with ET and PV and it might contribute to the pathogenesis of thromboembolic events in these patients.

Markers of Endothelial and Platelet Status in Patients with Essential Thrombocythemia and Polycythemia Vera.

Vascular complications are the main cause of morbidity in polycythemia vera (PV) and essential thrombocythemia (ET). To investigate plasma concentrations of soluble P-selectin (sP-Sel.), soluble E-selectin (sE-Sel.) and soluble thrombomodulin (sTM) in relation to the presence of thromboembolic events 38 patients with Chronic Myeloproliferative Disorders (CMD) (14 PV pts and 24 ET pts), 15 age-matched controls and 15 patients with secondary thrombocytosis were studied. Plasma levels of P-Sel., E-Sel. and TM were significantly increased in the group of patients as compared with control subjects (respectively p < 0.001, p < 0.04 and p < 0.01). sP-Sel. levels showed no significant difference between the patients and those with secondary thrombocytosis. No difference in sP-sel levels were also observed between subgroups of CMD patients with and without vascular complications. However, among patients with ET, those with thrombosis had higher sP-Sel levels than those without thrombosis (1.177 ± 110.48 ng/ml vs 816.25 ± 99.27 ng/ml). High levels of sE-Sel and sTM were found in CMD patients (71.93 ± 39.08 ng/ml and 35.81 ± 20.79 ng/ml, respectively). Plasma sE-Sel. concentration was significantly higher in CMD patients with thrombosis than that in CMD patients without thrombosis (p < 0.001). There was no difference in sTM concentration between two groups. These findings indicate that sustained endothelium and platelet activation is present in patients with ET and PV and it might contribute to the pathogenesis of thromboembolic events in these patients.

Increased levels of the soluble adhesion molecule E-selectin in patients with chronic myeloproliferative disorders and thromboembolic complications.

Patients with chronic myeloproliferative disorders (CMD) show a high frequency of thrombosis. For this reason we evaluated endothelial cell markers, soluble adhesion molecule E-selectin (sELAM), and thrombomodulin (TM) in 25 patients with CMD. Among them nine presented thromboses in their past history. Data were compared with those obtained in a group of healthy subjects and a group of patients with secondary thrombocytosis. The mean plasma concentrations of sELAM were elevated in patients with CMD, as compared with healthy subjects (81.27 +/- 42.8 ng/ml vs. 41.75 +/- 13; P < 0.02). Similarly, the mean plasma concentrations of sTM were increased in CMD patients in comparison with the control group (102.0 +/- 73 ng/ml vs. 16.7 +/- 9.6; P < 0.01). More markedly elevated sELAM levels were observed in CMD patients with thrombosis than in patients without thrombosis (113.16 +/- 29.5 ng/ml vs. 55.11 +/- 19.1 ng/ml; P < 0.001), while no significant difference was found between CMD patients without thrombosis and secondary thrombocytosis (50.72 +/- 10.8 ng/ml). Plasma thrombomodulin values in CMD patients with thrombosis (131 +/- 93.8 ng/ml) were higher than those without thrombosis (65.77 +/- 43.9 ng/ml; P < 0.02). sTM values were also significantly increased in patients with secondary thrombocytosis (P < 0.01). It is speculated that the plasma, sELAM levels may reflect endothelium activation and that it is possibly useful in predicting the thrombotic risk in myeloproliferative disorders.

Leukocyte integrin very late antigen-4/vascular cell adhesion molecule-1 adhesion pathway in splanchnic artery occlusion shock.

We investigated the role played by the very late antigen-4 (VLA-4)/ vascular cell adhesion molecule-1 (VCAM-1) interaction in the pathogenesis of splanchnic artery occlusion shock. Splanchnic artery occlusion shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min. Sham operated animals were used as controls. Survival time, serum tumour necrosis factor (TNF-alpha), monocyte and lymphocyte cell count and the responsiveness to acetylcholine of aortic rings were studied. Furthermore we investigated the VCAM-1 expression on vessel endothelium and the percentage of VLA-4 positive leukocytes. Splanchnic artery occlusion shocked rats had a decreased survival time (76 +/- 10 min, while sham shocked rats survived more than 4 h), increased serum levels of TNF-alpha (328 +/- 11 U/ml), a decreased number of both monocytes and lymphocytes and reduced responsiveness to acetylcholine (10 nM-10 microM) of aortic rings. In addition we found an increased expression of endothelial VCAM-1 on aortic rings and a reduced percentage of VLA-4 positive lymphocytes and monocytes. Passive immunization with specific antibodies raised against either VCAM-1 or VLA-4 (2 mg/kg, i.v., 3 h before splanchnic artery occlusion shock) increased survival, improved monocyte and lymphocyte count and restored the responsiveness of aortic rings to acetylcholine (P < 0.01). Finally, inhibition of TNF-alpha biosynthesis reversed the increased endothelial expression of VCAM-1 and the reduced percentage of integrin VLA-4 positive leukocytes. Our findings suggest that (i) VLA-4/VCAM-1 interaction has a role in the pathogenesis of circulatory shock; (ii) this interaction might be a target for new therapeutic approaches to the therapy of low-flow states.

Monocytes and lymphocytes as active participants in the pathogenesis of experimental shock.

We investigated the role played by monocytes and lymphocytes in the pathogenesis of experimental shock. Splanchnic artery occlusion (SAO) shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min followed by reperfusion. Sham operated animals were used as controls. SAO shocked rats had a decreased survival time (80 +/- 11 min, while sham shocked rats survived more than 4 h), increased serum (248 +/- 21 U/ml) and macrophage (145 +/- 15 U/ml) levels of TNF-alpha, enhanced myeloperoxidase (MPO) activity in the ileum (3.38 +/- 0.2 U x 10(-3)/g tissue), decreased number of monocytes, lymphocytes and neutrophils and a profound hypotension. In addition we found an increased expression of vascular cell adhesion molecule-1 (VCAM-1) on aortic endothelium and a reduced percentage of VLA-4 positive monocytes and lymphocytes. Inhibition of TNF-alpha synthesis, reversed the increased endothelial expression of VCAM-1, increased the percentage of integrin VLA-4 positive leukocytes and improved monocyte, lymphocyte and neutrophil count. Furthermore a passive immunization with specific antibodies raised against VCAM-1 (2 mg/kg, i.v. 3 h before SAO) increased survival, reduced MPO activity in the ileum (0.034 +/- 0.04 U x 10(-3)/g tissue) and improved mean arterial blood pressure. Our data suggest that monocytes and lymphocytes participate in the pathogenesis of splanchnic ischaemia-reperfusion injury and may amplify the adhesion of neutrophils to peripheral tissues.

Circulating levels of intercellular adhesion molecule-1 (ICAM-1) and soluble interleukin 2 receptors (IL-2R) in patients with B cell chronic lymphocytic leukaemia.

The serum concentrations of circulating ICAM-1 (cICAM-1) and soluble receptors for interleukin-2 (sIL-2R) were evaluated on 48 patients with B-cell chronic lymphocytic leukaemia (B-CLL) and on 15 healthy control subjects. The mean +/- SD concentration of cICAM-1 was significantly higher (p < 0.002) in B-CLL patients (407.7 +/- 164.3 ng/ml) than in healthy controls (245.4 +/- 76.7 ng/ml). Patients with progressive disease had higher cICAM-1 levels than patients with "indolent" disease (440.38 +/- 32.3 ng/ml versus 321.36 +/- 14.45 ng/ml; p < 0.0001). Serum levels of cICAM-1 were also significantly higher (p < 0.0002) in patients with advanced stage (III-IV) than in those with early stage (I-II). The increase of cICAM-1 levels was positively correlated to increases of soluble receptors for interleukin-2 (r = 0.9; p < 0.0001). These results seem to show that the measurement of serum levels of cICAM-1 may be an useful tool for monitoring disease activity and tumoral mass in patients with B-CLL. However, further studies are needed to define the functional role of high cICAM-1 levels in the immunological dysregulation of patients with malignancy.

Combined systemic beta-interferon and thymostimulin administration for metastatic brain tumors. A brief report.

Interferon-beta (IFN-B) plus thymostimulin (Th) were administered for eight weeks, intravenously and by intramuscular route to eight patients with metastatic brain tumors. No patient had a complete response. One had a stable disease and seven a progressive disease during that time. The mechanisms of the action of IFN-B and Th, and the possible therapeutic value are discussed.

Serum levels of 2',5 oligoadenylate synthetase during interferon therapy in patients with B-cell chronic lymphocytic leukemia.

We assayed 2',5 oligoadenylate synthetase (2'5-AS) activity in the serum of eight patients with B-cell chronic lymphocytic leukemia (B-CLL) who were undergoing therapy with alpha interferon (IFN). Mean pretreatment levels of 2'5-AS were normal, but upon treatment the levels rose to higher than normal values. Moreover, the degree of enzyme induction in serum was significantly higher in CLL responders (P < 0.003) whereas no difference was found in non-responders. The lack of serum 2'5-AS induction in non-responder patients might be related to unresponsiveness to treatment with IFN. These results seem to show that the measurement of serum levels of 2'5-AS activity may be a useful tool in monitoring IFN treatment.

Intracellular and serum levels of aldolase activity in B chronic lymphocytic leukemia.

Intracellular and serum activities of aldolase (ALS) were biochemically determined in lymphocyte subpopulations from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL). Aldolase activity was significantly lower in T cells of CLL than in normal T cells (2.9 +/- 1.5 vs. 4.7 +/- 2.1 Sigma Units (SU)/6 x 10(6) cells, p < 0.05). The aldolase activity also was significantly (p < 0.001) lower (3.1 +/- 1.9 SU/6 x 10(6) cells) in CLL B lymphocytes than in normal B lymphocytes (18.1 +/- 6.5 SU/6 x 10(6) cells). Moreover, the serum levels of ALS in all patients with B-CLL were higher than that in normal subjects (8.1 +/- 5.8 vs. 2.2 +/- 0.8 SU/ml, p < 0.02). Our findings demonstrate that T lymphocytes from patients with B-CLL display enzyme activity different from that of normal T cells. This may reflect the abnormal maturity of the residual T cell population in B-CLL.

E rosette formation in B cell chronic lymphocytic leukemia.

We describe a patient with B cell chronic lymphocytic leukemia whose B lymphocytes showed the capacity to form rosettes with sheep red blood cells during prolymphocytoid transformation of the disease. In this case, rosette formation was found to be related to the presence of the classic E rosette receptor since the leukemic cells were recognized by the OKT11 monoclonal antibody. This feature was not shown at diagnosis. Evaluation of this phenomenon is discussed.