John Dervenoulas

The role of microRNAs in normal and malignant hematopoiesis

MicroRNAs are small non‐coding RNAs that act at the post‐transcriptional level, regulating protein expression by repressing translation or destabilizing mRNA target. Because of their discovery, microRNAs have been associated with almost every normal cell function, including proliferation, differentiation and apoptosis. Several lines of evidence suggest that they have an important role in normal hematopoiesis as exemplified by the role of mir‐155 and mir‐150 in the differentiation of B and T lymphocytes, the suppressive role of mir‐221 and mir‐222 in erythroid differentiation, the inhibitory effect of mir‐181 on hematopoietic differentiation and the induction of myeloid differentiation by mir‐223. Moreover, they play a role both as oncogenes, probably by a variety of mechanisms, namely through elimination of tumor suppressor proteins, or as tumor suppressor genes by targeting oncogenic mRNAs. Their aberrant expression has been associated with solid tumors and hematopoietic malignancies as suggested by the frequent deletion of mir‐15a and mir‐16‐1 in chronic lymphocytic leukemia, the increased levels of mir‐155 in diffuse large B‐cell lymphomas and the increased levels of mir‐181 in acute myeloid leukemia M1 and M2. The purpose of this review is to summarize current knowledge on the role of microRNAs in normal hematopoiesis and hematopoietic malignancies and, moreover, to highlight their role as potential therapeutic tools.

Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone with or Without Radiotherapy in Primary Mediastinal Large B-Cell Lymphoma: The Emerging Standard of Care

UNLABELLED More aggressive treatment approaches (methotrexate, cytarabine, cyclophosphamide, vincristine, prednisone, and bleomycin [the MACOP-B regimen] or consolidation with high-dose therapy and autologous stem cell transplantation) have been considered to be superior to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) in patients with primary mediastinal large B-cell lymphoma (PMLBCL). Rituximab-CHOP (R-CHOP) is the standard of care for diffuse large B-cell lymphoma, whereas efficacy in PMLBCL has not been adequately confirmed. PATIENT AND METHODS Seventy-six consecutive PMLBCL patients who received R-CHOP with or without radiotherapy (RT) were compared with 45 consecutive historical controls treated with CHOP with or without RT. Baseline characteristics of the two groups were balanced. RESULTS The rate of early treatment failure was much lower with R-CHOP with or without RT (9% versus 30%; p = .004). The 5-year freedom from progression rate after R-CHOP with or without RT was 81%, versus 48% for CHOP with or without RT (p < .0001). The 5-year event-free survival rates were 80% and 47% (p < .0001) and the 5-year overall and lymphoma-specific survival rates were 89% and 69% (p = .003) and 91% and 69% (p = .001), respectively, with only seven of 76 lymphoma-related deaths. Among R-CHOP responders, 52 of 68 received RT. CONCLUSIONS Based on these results, most patients with PMLBCL appear to be cured by R-CHOP in 21-day cycles with or without RT, which could be the current standard of care. Therefore, the need for more aggressive treatment strategies is questionable unless high-risk patients are adequately defined. Further studies are required to establish the precise role of RT.

Thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS): treatment outcome, relapses, prognostic factors. A single-center experience of 48 cases.

Abstract The thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) is a rare disorder characterized by microangiopathic hemolysis and thrombocytopenia. We have undertaken a retrospective analysis of the clinical characteristics, treatment outcome, and prognosis of 48 patients diagnosed and treated in our institution during a 13-year period. Among our patients 22 (46%) had fever, 35 (73%) neurological abnormalities, and 22 (46%) renal impairment at presentation of the syndrome. All patients were treated with a multimodality regimen including plasma exchange, steroids, antiplatelet agents, and IgG infusion. Of the 48 patients, 41 achieved complete remission, two had a partial response, and five had no response and died of progressive disease. Within a median follow-up period of 40 months, ten of the 41 patients who had achieved remission relapsed, most of them within the first 2 years, and nine of these responded promptly to plasma exchange therapy. Eight deaths were observed, seven of refractory disease and one in fourth relapse. The analysis of prognostic factors revealed advanced age and severe renal impairment (creatinine levels above 2 mg%) as the only parameters associated with treatment failure and poor outcome. However, none of the pretreatment characteristics proved to be of prognostic value regarding the probability of relapse. In conclusion, TTP/HUS represent a syndrome of variable clinical expression and aggressiveness. The use of a multimodality regimen in our series produced a high response rate. Nevertheless, the early identification, based on clinical characteristics, of poor-prognosis cases that probably need more or alternative forms of treatment is an issue that remains to be elucidated in prospective trials.

Myelodysplastic syndromes: Analysis of 131 cases according to the FAB classification

The clinical and haematological findings in 131 patients with myelodysplastic syndromes (MDS), none of which had previously received chemotherapy or radiotherapy, classified according to the FAB criteria, were analysed. The distribution among the 5 subgroups was: RA 31 patients, RAS 19, RAEB 23, CMML 29 and RAEBT 29 patients. There were difficulties in the classification of 24 patients. These included, first, 8 cases with myeloid hyperplasia of the bone marrow (BM) but without mono‐cytosis or excess of blasts of the BM. They were classified as RA. Second, 8 cases with sideroblastosis but with monocytosis or excess of blasts of the BM were classified 3 as RAEB, 2 as CMML and 3 as RAEBT. Finally, 8 cases with absolute monocytosis and BM blasts 15–30% were classified as CMML. 37 of 82 dead patients (45.1%) had transformed to acute non‐lymphoblastic leukaemia (ANLL). The incidence of evolution to ANLL was low for RA and RAS (6.30% and 12.5% respectively), while it was 37.5% for RAEB, 57.1% for CMML and 77.2% for RAEBT. The median survival for each subgroup was: RA 18 months; RAS 25; RAEB 13; CMML 14 and RAEBT 10 months. It is concluded that the FAB classification with some modifications recognises group of MDS with different prognosis.

Primary extranodal non-Hodgkin's lymphoma of the head and neck

Primary extranodal NHL of the head and neck (HN-NHL) accounts for 10-20% of all cases of NHL. Despite their frequency, the natural history and biological behaviour of these lymphomas is poorly understood. In this study we analysed the data 116 cases of HN-NHL. There were 65 males and 51 females with a median age of 56 years. The distribution among different anatomical sites was: tonsils 56 cases (48.3%), nasopharynx 15 (12.9%), mandible/gingiva 9 (7.8%), hard palate 7 (6%), parotis 6 (5.2%), nasal cavity 6 (5.2%), hypopharynx/larynx 6 (5.2%), thyroid 5 (4.3%), ocular adnexa 4 (3.5%), paranasal sinuses 2 (1.7%). The patients were treated with radiotherapy alone (14 cases), combined chemotherapy (52 cases) and combined modality (50 cases). According to the WF histological classification 73 cases (62.9%) had intermediate, 32 (27.6%) high and 11 (9.5%) low grade. Patients were separated in two groups: Tonsillar NHL (56 cases) and NHL of all other sites (non-tonsillar group-60 cases). A comparison between the two groups showed that there was no statistically significant difference with respect to age, sex, and histological subtypes. Also treatment response was similar (82.1% for the tonsillar vs 83.3% for the non-tonsillar). The two groups differed in stage distribution, survival and pattern of relapse. Stage I was more frequent in the non-tonsillar NHL (60%) in contrast to tonsillar NHL where stage II was more prominent (51.8%). Median survival was 86 months for the tonsillar while it has not been reached yet for the non-tonsillar patients. Patients in stage I and stage II of the non-tonsillar group had better survival compared to stages I and II of the tonsillar patients. Finally GI tract was a common site of relapse in the tonsillar group while a considerable number in CNS relapses were observed in the non-tonsillar group. We concluded that HN-NHL constitutes a heterogeneous group of patients. Tonsillar lymphomas represent a distinct group with some special clinicopathological findings.

Extracorporeal photopheresis in combination with bexarotene in the treatment of mycosis fungoides and Sézary syndrome

were histologically compatible with MDE. Elastica staining showed band-like and sharply demarcated elastolysis in the upper reticular dermis with conservation of the fine vertically orientated elastic fibres of the papillary dermis. In addition, focal areas of fragmented and absent elastic fibres were observed in the mid-reticular dermis (Fig. 2c). Only a scant perivascular lymphohistiocytic infiltrate was seen in the upper dermis. On closer examination, isolated and small clusters of CD68 (PGM1)-positive multinucleated giant cells were detected in the mid-reticular dermis showing elastophagocytosis. This case documents the transition of AEGCG into a noninflammatory end stage, clinically and histologically compatible with MDE. The pathogenesis of MDE is still unknown. Exposure to natural or artificial UV radiation frequently precedes the onset of MDE suggesting a pathogenic UV-mediated immunological mechanism. However, MDE lesions are not usually confined to sun-exposed areas, so that other cofactors are likely to play a role. Extensive sun exposure was denied by our patient. An association of MDE with nicotine abuse and oral contraceptives as in our case has repeatedly been reported. It is still unclear if this is mere coincidence or if these substances represent relevant trigger factors. MDE has been described after resolution of urticaria, erythematous skin changes and lesions resembling granuloma annulare. In the majority of cases, however, MDE develops without a clinically obvious inflammatory prodromal stage. The clinical course in our patient suggests that AEGCG and MDE might represent different stages in the clinical spectrum of dermal elastolysis. After the inflammatory prodromal stage of AEGCG and phagocytosis of elastic fibres by multinucleate giant cells the noninflammatory ‘burned-out’ end stage of MDE could result in a loss of elastic fibres in the mid-dermis. As elastophagocytosis by macrophages is, to a much lesser extent, also a feature of early inflammatory MDE lesions, AEGCG might represent a severe inflammatory variant of the same entity.

Antiphospholipid syndrome: a predisposing factor for early onset HELLP syndrome

Hemolysis elevated liver enzymes low platelets syndrome (HELLP) is a relatively rare pregnancy-related thrombotic microangiopathic disorder, usually observed during the third trimester. Its incidence seems to be increased in patients with antiphospholipid syndrome (APS). In this report, we describe a 33-year-old pregnant woman with previously known primary APS who developed early onset HELLP syndrome during the 15th week of gestation. We also review the literature about this interesting relationship between APS and HELLP.

The Novel Member of the BCL2 Gene Family, BCL2L12, Is Substantially Elevated in Chronic Lymphocytic Leukemia Patients, Supporting Its Value As a Significant Biomarker

BCL2L12 is a recently identified gene belonging to the BCL2 family, members of which are implicated in hematologic malignancies, including chronic lymphocytic leukemia (CLL). The aim of this study was to analyze the mRNA expression of the novel apoptosis-related gene BCL2L12 in patients with CLL and to examine its prognostic and predictive value and potential clinical application as a novel molecular biomarker for CLL. For this purpose, total RNA was isolated from peripheral blood of 65 CLL patients and 23 healthy donors. An ultrasensitive quantitative real-time polymerase chain reaction methodology for BCL2L12 and BCL2 mRNA quantification was developed using SYBR Green chemistry. After preparing cDNA by reverse transcription, relative quantification analysis was performed using the comparative C(T) (2(-ΔΔCT)) method. Furthermore, analysis of IGHV mutational status, CD38 expression, and detection of early apoptosis by double staining with Annexin V-FITC and propidium iodide were performed. According to our findings, BCL2L12 mRNA expression is significantly higher in CLL patients than in healthy donors. Receiver operating characteristic analysis demonstrated that BCL2L12 expression had significant discriminatory value, distinguishing very efficiently CLL patients from the non-leukemic population. Moreover, BCL2L12 expression predicts the presence of CLL, as demonstrated by both univariate and multivariate logistic regression analyses. Finally, high BCL2L12 mRNA levels are associated with advanced clinical stage and predict shorter overall survival in CLL patients.

Efficacy of Intravenous Immunoglobulin in the Treatment of Thrombotic Thrombocytopaenic Purpura

Thrombotic thrombocytopaenic purpura (TTP) is characterised by platelet aggregation in the capillaries, thrombocytopaenia and microangiopathic haemolytic anaemia that result in organ ischaemia, mainly of the CNS and kidneys. Since the institution of plasma exchange therapy no further treatments have been proved to improve the survival and the relapse rate of TTP patients. In this retrospective study, we evaluated the efficacy of normal human immunoglobulin treatment in 44 patients suffering from TTP. Patients were divided into two groups that either did not receive (group A: 15 patients) or received (group B: 29 patients) 400 mg/kg of human normal immunoglobulin intravenously (ivIgG) for 5 days. All patients received treatment with corticosteroids, anti-platelet agents and plasma exchange. The results clearly showed that there was no statistically significant difference between the two groups in either remission rate or time to relapse following remission. In conclusion, this study did not prove any beneficial effect of ivIgG in the treatment of TTP patients.

Mutational and methylation analysis of the cyclin-dependent kinase 4 inhibitor (p16INK4A) gene in chronic lymphocytic leukemia.

Abstract:  Objectives: Chronic lymphocytic leukemia (CLL) comprises a heterogenous group of at least two types of disease entities characterized by distinctive clinical, immunophenotypical and genetic features. The molecular mechanisms underlying the pathogenesis and the histological transformation of CLL are not well known. The INK4A/p16, a cyclin dependent kinase inhibitor has been considered as a tumor suppressor gene. Inactivation of this gene by homozygous deletions, mutations and hypermethylation occurs in a variety of human neoplasms. The aim of the present study was to determine the frequency of p16 gene deletions and mutations as well as the methylation status of the same gene in CLL patients. Methods: We examined 34 samples from CLL patients by Southern Blotting, Single‐Strand Conformation Polymorphism (SSCP), DNA sequencing and Methylation‐Specific PCR. Results: Southern Blot analysis revealed non‐rearranged bands in 33/34 cases. Homozygous deletions were not observed in any case. In 1/34 case a rearranged band was detected with EcoRI enzyme. The PCR‐SSCP analysis of exons 1 and 3 revealed normal pattern of migration in all cases examined. The analysis of exon 2 revealed abnormal migration pattern in 2/34 cases (5.8%). Sequencing of these cases revealed the presence of the ALA148THR polymorphism. Methylation analysis of p16 gene promoter revealed hypermethylation of CpG islands in 6/34 cases (17.6%). Conclusion: These results indicate that genetic alterations of p16 gene are rare events in patients with CLL. The clarification of the role of p16 gene promoter methylation in the pathogenesis and evolution of CLL needs further investigation.