Incidence, risk factors, and outcome of asymptomatic central nervous system involvement in adult patients with acute myeloid leukemia.

Examination of central nervous system (CNS) involvement is not routine diagnostic practice in adult patients with acute myeloid leukemia (AML). Therefore, many asymptomatic patients with CNS involvement might go undetected. The effect of CNS involvement on the AML disease course is not well defined, with conflicting results regarding clinical outcome. This study aimed to determine the incidence of asymptomatic CNS involvement in AML estimated by multiparametric flow cytometry of cerebrospinal fluid (MFC-CSF) at diagnosis, the related potential risk factors, and prognosis. In total, 645 patients with de novo AML were screened; 183 (28.4%) of them fulfilled institutional practice for MFC-CSF analysis based on presence of CNS symptoms and/or clinical features. CNS symptoms and signs were observed in 8/183 (4.4%) patients, but most patients (175/183, 95.6%) were asymptomatic. In the asymptomatic group, 73/175 (41.7%) patients had positive or suspicious cerebrospinal fluid (CSF) findings categorized as CNS positive (CNSpos) and 102/175 (58.3%) had normal CNS findings categorized as CNS negative (CNSneg). The presence of leukemic blasts was confirmed in 81/183 (44.3%) patients; the total incidence of CNS involvement in the whole AML group was 12.6% (81/645). Compared with asymptomatic patients with CNSneg, those with CNSpos had a significantly higher frequency of lymphadenopathy, white blood cell count ≥30 × 109/L, presence of the monocytic phenotype, and a high percentage of bone marrow (BM) blasts. The multivariate logistic regression model identified monocytic phenotype (p = 0.047) and high percentage of BM blasts (p = 0.042) as predictors for CNSpos. CNSpos did not affect overall survival in patients with AML. There was a higher incidence of CNS involvement in asymptomatic adult patients with de novo AML, emphasizing possible undervalued rates of CNS disease at diagnosis. Prospective studies should determine whether diagnostic lumbar puncture for MFC-CSF analysis and CNS prophylaxis could contribute to better selection and prognosis in this patient population.

Gene expression profile of circulating CD34(+) cells and granulocytes in chronic myeloid leukemia.

PURPOSE: We compared the gene expression profile of peripheral blood CD34(+) cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR-ABL oncogene. METHODS: The microarray analyses have been performed in circulating CD34(+) cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR-ABL positive CML patients were in chronic phase, with a mean value of 2012±SD of CD34(+)cells/µl in peripheral blood. RESULTS: The gene expression profile was more prominent in CML CD34(+) cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34(+) cells (HINT1, TXN, SERBP1) and granulocytes, respectively. BCR-ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11, CDK4/6, and MYC and reduction of E2F1, KRAS, and NFKBIA gene expression in CD34(+) cells. Among genes linked to the inhibition of cellular proliferation by BCR-ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. CONCLUSION: The presence of BCR-ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34(+) cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects.

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms.

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34(+) cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34(+) cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs.

Correlation between leukocyte-platelet aggregates and thrombosis in myeloproliferative neoplasms.

INTRODUCTION: The impact of activated blood and endothelial cells on the thrombosis in myeloproliferative neoplasms (MPN) has not yet been clarified. We prospectively analyzed correlation between circulating leukocyte-platelet aggregates and soluble selectins to thrombosis occurrence in MPN, in the context of standard and cardiovascular risk factors, and different clinical and biological characteristics. METHODS: Flow cytometric analysis of neutrophil-platelet (Neu-Plt) and monocyte-platelet (Mo-Plt) aggregates in peripheral blood, as well as quantification of soluble E-/L-/P-selectins by enzyme immunoassay, was performed on 95 newly diagnosed MPN patients. RESULTS: During the follow-up, thrombosis occurred in 12.6% MPN patients (arterial 9.4%, venous 3.2%), with a mean time of 39 months. The overall incidence rate of main thrombotic events was 4.36 per 100 patient-years. The incidence of arterial hypertension (HTA) was significantly higher in patients with thrombosis, compared to those without thrombosis (P < .05). The level of soluble P-selectin was significantly higher in patients with thrombosis compared to those without thrombosis (346.89 ng/mL vs 286.39 ng/mL, P = .034). The mean level of Neu-Plt (26.7% vs 22.4%) and Mo-Plt (17.8% vs 12.3%) aggregates did not differ significantly between the groups with and without thrombosis. A multivariate COX proportional hazard regression model confirmed an independent predictive significance of Mo-Plt aggregates (HR = 1.561, 95% CI: 1.007-2.420, P = .046), as well as the cumulative effect of Mo-Plt aggregates and HTA (HR = 1.975, 95%CI: 1.215-3.212, P = .006) for thrombosis occurrence. CONCLUSION: Monocyte-platelet aggregates represent an independent risk factor for thrombosis occurrence, further on supported by HTA.

CD56-Positive Acute Myeloid Leukemia Following Treatment of Hairy Cell Leukemia with Cladribine - Report of 2 Cases and Review of the Literature.

Treatment of hairy cell leukemia (HCL) with alfa-interferon and purine analogs significantly prolongs survival in these patients. However, with life prolongation, an increased risk of secondary malignancies has been reported. Acute myeloid leukemia (AML), as a second malignancy after HCL treatment is extremely rare and has been reported in only 12 cases so far. We here report additional 2 cases of CD56+ AML developed after sustained clinical remission of HCL achieved with cladribine (2 and 6 years after, respectively). The first patient refused chemotherapy and shortly thereafter died. The second patient responded to chemotherapy and was successfully allo-transplanted. Three years later, the patient is in stable clinical remission, which is a unique case in the literature. In conclusion, it is not clear whether development of AML in HCL patients is caused by mutagenic potential of the applied chemotherapy or by immune suppression/ perturbations as a characteristic of the underlying disease.

The predictive value of morphological findings in early diagnosis of acute myeloid leukemia with recurrent cytogenetic abnormalities.

This study explores cytomorphologic features and their predictive role for early identification of acute myeloid leukemia (AML) with morphological distinctive recurrent cytogenetic abnormalities (RCA): t(15;17), t(8;21) and inv(16)/t(16;16). We retrospectively evaluated 396 de novo AML cases, diagnosed and treated at single institution, between 2013-2017. Specific cytomorphologic features suggesting distinctive AML-RCA were revealed at diagnosis in 62 (15.65%) patients, including AML with t(15;17) in 41 (66.13%), t(8;21) in 13 (20.97%) and inv(16)/t(16;16) in 8 (12.90%). Final diagnoses of AML-RCA according to WHO integrated diagnostic criteria were established in 66 (16.66%) cases, including AML with t(15;17) 40 (60.60%), t(8;21) 17 (25.76%), and inv(16)/t(16;16) 9 (13.64%). Discordance between cytomorphological and other integrated criteria was detected as missed/wrong-call in 0/1 for t(15;17), 6/2 for t(8;21) and 2/1 for inv(16)/t(16;16). The cytomorphological accuracy was 97.56% (40/41) for t(15;17), 57.89% (11/19) for t(8;21) and 70% (7/10) for inv (16)/t(16;16). Positive/negative predictive values of cytomorphological evaluation were: 97.56%/100% for t(15;17); 84.62%/88.68% for t(8;21); 87.50%/96.65% for inv(16)/t(16;16). Sensitivity/specificity were: 100%/96.15% for t(15;17); 64.10%/95.92% for t(8;21); 77.78%/98.25% for inv(16)/t(16;16). We confirmed that morphology is still a highly relevant evaluation method in diagnosing several common AML-RCAs before completing cytogenetic and molecular studies, enabling early detection, particularly of AML with t(15;17).

Good's syndrome with increasing γδ T-lymphocyte subpopulation: A case report.

INTRODUCTION: Good's syndrome is a rare cause of adult-onset immunodeficiency associated with thymoma. Good's syndrome should be considered in patients older than 40 years with the history of frequent infections. An abnormal immunoglobulin profile needs further investigation and flow cytometry which is crucial for establishing the diagno- sis of Good's syndrome. CASE REPORT: We present a 56- year-old men with Good's syndrome diagnosed after a two-year history of recurrent infections. Examination of immune status of the patient showed decreased serum levels of all immunoglobulins. Flow cytometry of peripheral blood lym-phocyte revealed markedly reduced peripheral B cells, CD4 T-cell lymphopenia, inverted CD4/CD8 T-cell-ratio 0.37 (CD4--20.82%, CD8--70.7%). Analysis of the subpopulations of T-lymphocytes showed relative increasing γδ T cell receptor (TCR) T lymphocytes. Computed tomography scan of the chest showed a mediastinal mass compatible with thymoma of the diameter of 40 mm. After initiation of intravenous immunoglobulins the patient was in the good clinical condition and without bacterial complications. As the patient refused the operative treatment we continued to control the mediastinal tumor mass which did not increase during a 3-year follow-up. CONCLUSION: The presented patient had a typical immunological finding for Good's syndrome, but also the increase in γδ TCR T-lymphocyte subpopulation for which it is difficult to determine whether this is pathogenetic or secondary reactive event.

The emergence of non-secretory multiple myeloma during the non-cytotoxic treatment of essential thrombocythemia: a case report.

INTRODUCTION: The emergence of multiple myeloma as a second malignancy in patients with essential thrombocythemia is extremely rare. Several cases have been published so far, pointing out the impact of a cytotoxic effect during treatment of essential thrombocythemia on the development of multiple myeloma. CASE PRESENTATION: We report the case of a 52-year-old Caucasian man who presented to our hospital because of leukocytosis, a slightly decreased hemoglobin level and thrombocytosis. After a complete hematological work-up, essential thrombocythemia was diagnosed. The patient was included in a multicenter clinical study, treated with anagrelide and his platelet counts were maintained in the normal range for more than 3 years. A sudden drop in his hemoglobin level with normal leukocyte and platelet count occurred at the same time as a back pain. Magnetic resonance imaging of his spine revealed the existence of a pathological fracture of Th4, the collapse of the upper edge of Th7 and osteolytic lesions of multiple thoracic vertebrae. Repeated hematological examinations, including bone biopsy with immunohistochemistry, disclosed diagnosis of multiple myeloma of the non-secretory type. CONCLUSIONS: To the best of our knowledge this is the first published case in which multiple myeloma developed during the treatment of essential thrombocythemia with the non-cytotoxic drug anagrelide. Our attempts to find a common origin for the coexistence of multiple myeloma and essential thrombocythemia have not confirmed the genetic basis of their appearance. Further studies are needed to determine the biological impact of this coexistence.

Prognostic significance of CD56 antigen expression in patients with acute myeloid leukemia.

The aims of this study were to investigate the frequency and prognostic relevance of CD56 expression in patients with acute myeloid leukemia (AML) and to compare the importance of CD56 expression with standard prognostic factors, such as age, leukocytosis, cytogenetic abnormalities and performance status. We analyzed the data of 184 newly diagnosed patients with non-promyelocytic AML and a follow-up of 36 months. The median patient age was 58 years, with a range of 18-79. CD56+ antigen was recorded in 40 patients (21.7%). CD56 + was the most significant risk factor for OS: P = 0.05. The most significant factor for a poor rate of CR was age ≥ 55 years (P = 0.001). CD56 positivity had no significant influence on CR rate, but it was the most significant risk factor for disease-free survival (P = 0.005). The CD56 antigen is an independent prognostic risk factor, and its presence should be measured regularly for a better prognostic assessment of patients with AML.

Prognostic factors for therapy-related acute myeloid leukaemia (t-AML)--a single centre experience.

Prognostic parameters for treatment outcome in 42 consecutive patients with t-AML diagnosed and treated in a single centre between 2000-2010 (mean age: 56.07 years, range: 23-84; 30 females) were evaluated retrospectively/prospectively. Antecedent malignancy occurred in 37 patients (88.15%): 28 solid cancers (breast, n=14), nine haematological. History of previous chemotherapy (CT), radiotherapy (RT) alone and combined CT/RT was present in 42.9%, 6.19% and 30.1% patients, respectively. Primary disease was active in 11 patients (six relapsed or metastatic cancers; five autoimmune diseases). Myelodysplastic syndrome preceded t-AML in 29% of patients. Median latency period from prior CT/RT was 54.62 months (range: 6-243). Median WBC count was 27.23 × 109/L, platelet count 62.29 × 109/L, haemoglobin level 87.83 g/L, peripheral blood and bone marrow blast percentage 30.7% and 66.7% respectively, serum LDH 1216 U/L. Aberrant expression of B or T lymphoid markers was registered in seven out of 39 and six out of 39 patients, respectively. Aberrant karyotype was detected in 24 out of 33 (72.7%) of eligible patients: favourable: 15.2%, intermediate: 42.4% and unfavourable: 42.4%. Eastern Cooperative Oncology Group (ECOG) performance status greater or equal to 2 and Haematopoietic Cell Transplantation Specific Comorbidity Index (HCT-CI) greater or equal to 3 exhibited 83.3% and 76.2% patients, respectively. Intensive induction CT for t-AML was administered in 24 patients. The median follow-up and the median overall survival (OS) for the whole cohort were 2 months and 5.94 months (range: 0.5-34), respectively. In 10 patients (23.8%) achieving complete remission (CR), median disease free survival (DFS) was 11.8 months (range: 4-32). Only CD19 expression, pretreatment karyotype, ECOG PS, HCT-CI and activity of primary disease had impact on OS (P<0.05).

Therapy-related acute leukemia in two patients with multiple sclerosis treated with Mitoxantrone.

Two cases of therapy-related acute leukemia (TRAL) after the use of Mitoxantrone for the treatment of secondary progressive multiple sclerosis (MS) are reported. They were extracted from the group of 42 consecutive patients with TRAL diagnosed and treated in single centre between 2000-2010. They were the only two with MS and the only two treated with Mitoxantrone. The first patient was a 43-year-old male with a previous history of MS of 15-year-duration, who developed acute promyelocytic leukemia 9 months following Mitoxantrone therapy (cumulative dose 120 mg). The second patient was a 55-year-old female suffering from MS for 16 years, who developed acute mixed-phenotype leukemia, T/myeloid type, with 46,XX,del(7)(p13)[12]/47,XX,idem,+3/[6]/46,XX[2], 15 months after completion of Mitoxantrone therapy (cumulative dose 100mg). Acute mixed-phenotype leukemia, T/myeloid type is for the first time described in the context of prior Mitoxantrone therapy. Although the incidence of TRAL in relation to Mitoxantrone pretreatment is rare, we should be vigilant for the prompt identification of this adverse event.

Concomitant aberrant methylation of p15 and MGMT genes in acute myeloid leukemia: association with a particular immunophenotype of blast cells.

In this study, methylation-specific polymerase chain reaction (MS-PCR) was used to define the methylation status of the target promoter sequences of p15 and MGMT genes in the group of 21 adult patients with acute myeloid leukemia (AML). The incidence of aberrant hypermethylation of p15 gene (71 %) was higher comparing to MGMT gene (33 %), whereas concomitant methylation of both genes had 24 % of the patients. Although the incidence of cytogenetic abnormalities between the groups with a different methylation status of p15 and/or MGMT genes was not significantly different, we observed general trend of clustering of abnormalities with adverse prognosis into groups with concomitant hypermethylation of both genes and only p15 gene. Also, we showed that AML patients with concomitant methylation of p15/MGMT genes had a higher proportion of leukemic blast cells characterized with specific expression of individual leukocyte surface antigens (CD117(+)/CD7(+)/CD34(+)/CD15(-)), indicating leukemic cells as early myeloid progenitors. Although we could not prove that hypermethylation of p15 and/or MGMT genes is predictive parameter for response to therapy and overall survival, we noticed that AML patients with comethylated p15/MGMT genes or methylated p15 gene exhibited a higher frequency of early death, lower frequency of complete remissions as well as a trend for shorter overall survival. Assessing of the methylation status of p15 and MGMT genes may allow stratification of patients with AML into distinct groups with potentially different prognosis.

[Prognostic significance of new biological markers in chronic lymphocytic leukaemia].

INTRODUCTION: Chronic lymphocytic leukaemia is a disease with heterogeneous clinical course and outcome. Some patients have a progressive course of the disease and require therapy immediately after the diagnosis, while others have a stable form without the need for treatment. Recently, two new biological markers, the expression of CD38 antigen and ZAP-70 have shown independent significance in the prognosis in CLL patients. OBJECTIVE: The aim of our study was to evaluate the clinical value of CD38 antigen and ZAP-70 expression as predictors of the disease progression and to analyze the correlation of these markers with other B-CLL prognostic markers. METHODS: We assessed the expression of CD38 antigens by flow cytometry on peripheral blood samples and the expression of ZAP-70 by immunohistochemistry on formalin-fixed bone marrow (BM) biopsies in 40 newly diagnosed B-CLL patients. Disease progression was defined by the period elapsed from diagnosis to the time to first treatment (TFT). RESULTS: Expression of CD38 antigen correlated positively with ZAP-70 expression (Pearson, r = 0.476; p = 0.002). Also, correlation analysis results showed that a positive expression of CD38 and ZAP-70 statistically significantly correlated with unfavourable classical prognostic parameters, such as advanced Binet stage C, diffuse BM infiltration, increased lactate-dehydrogenase and beta-2 microglobulin serum levels. Patients with positive expression of CD38 antigen and ZAP-70 had a shorter TFT (log rank, 0.003 vs. 0.049). CONCLUSION: Both new biological markers were shown to have an exceptional significance in the prediction of prognosis in CLL patients.