Chronic lymphocytic leukemia: assessing pathogenesis and prognosis by modern molecular cytogenetic studies and microRNAs expression

Chronic lymphocytic leukemia (CLL) is a B-cell clonal lymphoprolipherative disorder characterized by the accumulation of small lymphocytes in the peripheral blood, bone marrow and lymph nodes deriving from the transformation of CD5+ B-cell. Despite a homogeneous immunophenotype consisting of CD19+, CD20+, CD5+ and CD23+, CLL is clinically heterogeneous. Several adverse prognostic features have been identified including stage, CD38 positivity, the unmutated configuration of the variable region of the immunoglobulin heavy chain gene (IGHV), ZAP70 positivity, chromosome aberrations and molecular abnormalities. Detailed immunophenotypic and genetic analysis allowed for the identification of a number of markers of activation and genetic instability, some of which are gaining relevance in clinical practice to predict outcome. Cell surface CD38 is one of these markers since it is an indicator of cell activation and proliferation that may prelude clonal evolution and worse clinical outcome. We therefore studied the biological and clinical significance of the presence of genetic heterogeneity in the minor CD38+ leukemic population, in a cohort of untreated low-risk CD38-negative CLL patients, defined by the presence of <7% CD38+ cells, and by the absence of unfavourable genetic lesions. Our data showed that a significant proportion of CD38- CLL patients with low risk FISH findings presented genetic aberrations within CD38+ cells. Most of these abnormalities were high risk lesions (11q deletion and 17p deletion) and, in most of the cases, these lesions were found in different cells indicating that multiple cytogenetically unrelated minor clones may be present in the CD38+ cell fraction. Interestingly, the presence of these additional FISH lesions in the small CD38+ cell fraction was associated with shorter time to first treatment (TTT). To identify biomarkers associated with this phenomenon, we performed miRNA expression analysis. We were thus able to show a deregulated miRNA expression profile in CLL cases with additional FISH lesions in CD38+ cells. In particular, miR-125a-5p was found to be downregulated both in CD38+ and CD38- cells in patients with FISH abnormal clones as compared to patients without FISH abnormal clones. The relevance of miR-125a-5p as a biomarker of inferior outcome and genetic complexity was then validated in a prospective cohort. In this validation cohort, we were able to confirm the predictive role of miR-125a-5p down-regulation in terms of shorter TTT. In addition we found that CLL patients with lower levels of miR-125a-5p displayed an increased rate of mutations in CLL-related genes by next-generation sequencing. Several recent studies have shown that CD38 expression is higher in CLL cells in the bone marrow and lymphoid tissues, especially in the proliferation centres (PCs), which are regarded as the histologic hallmark of this disease. Indeed CLL is a disease in which the host’s microenvironment promotes leukemic cell growth, leading to sequential acquisition and accumulation of genetic alterations and proliferation centers may play an important role in the biology of CLL, as they represent its proliferative compartment. To better define the significance of proliferation centers, we studied lymph node biopsies taken from a cohort of patients by fluorescence in situ hybridization (FISH) studies using a 5-probe panel on tissue microarrays (TMA). The cases were classified into two categories: “PCsrich” and “typical”. The PCs-rich group was associated with 17p-, 14q32/IGH translocations and +12. The median survival from the time of TMA for PCs-rich and typical groups was 11 and 64 months respectively. The PCs-rich pattern was the only predictive factor of an inferior survival. These findings establish an association between cytogenetic profile and the amount of PCs in CLL, and show that this histopathologic characteristic is of value for risk assessment in patients with clinically significant adenopathy. CLL turned out to be a disease with multiple facets in its pathogenic mechanisms including genetic aberrations, antigen drive and microenvironmental interactions. In the first part of this work, we focused our attention on the correlation between CD38-positivity, proliferation centres and development of genetic aberrations. To translate this knowledge in clinical practice we planned further studies focusing i) on the correlation between chromosomal aberrations and clonal evolution and ii) on how to stratify patients into different risk-groups at diagnosis according to cytogenetic abnormalities and gene mutations. The presence of cytogenetic abnormalities is a hallmark of CLL. It was reported that a fraction of CLL patients developed new cytogenetic abnormalities at chromosome sites of known prognostic importance during the course of their disease (clonal evolution, CE). To better define the incidence and signature of CE, a cohort of patients were analysed sequentially by FISH. Recurring aberrations at clonal evolution were 14q32/IGH translocation, 17p-, 11q-, 13q- and 14q32 deletion. The development of CE occurred only in previously treated patients. Our data show that the 14q32/IGH translocation may represent one of the most frequent aberrations acquired during the natural history of CLL. CE occurs in pre-treated patients with short TTT and survival, after the development of CE with and without 14q32 translocation, is relatively short. Having assessed the incidence of chromosome aberration in CLL with evolution and/or adenopathy we next moved to CLL with an apparently “favourable” profile of cytogenetic lesions, to establish if improved cytogenetic techniques could help refine prognostication. We therefore designed a study to assess whether karyotypic aberrations in patients without FISH anomalies correlate with established clinical and prognostic parameters. The clinical and prognostic significance of karyotypic aberrations in normal FISH CLL was first evaluated in a retrospective single centre series of patients and then validated prospectively in a multicentre series of cases diagnosed and analysed for karyotype with DPS30/IL2 stimulation. Conventional karyotyping using DSP30/IL2 stimulation is an effective method for the detection of chromosome aberrations in approximately one third of CLL with normal FISH. The abnormal karyotype correlated with shorter time to first treatment and shorter survival. This set of data also showed that, in CLL patients with normal FISH, conventional cytogenetic analysis identifies a subset of cases with adverse clinical and prognostic features to be considered for the design of risk-adapted treatment strategies. In the last part of our experimental work we moved from the consideration that the cytogenetic lesions do not entirely explain the molecular pathogenesis and the clinical heterogeneity of CLL. Indeed, the advent of next‐generation sequencing (NGS) technologies has enabled exploration of the CLL genome, uncovering genetic lesions that recurrently target the leukemic cells. NGS studies have further elucidated the genomic complexity of CLL. In order to improve understanding of genetic basis of CLL and to apply NGS to CLL, we sequenced DNA samples from untreated patients affected by chronic lymphocytic leukemia with a panel of 20 genes and we correlated mutational status with clinicobiological parameters.Mutations were identified in the following genes: TP53, SF3B1, POT1 , ATM , MYD88 , FBXW7 , MAPK1 , DDX3X , KLHL6 , KRAS. The presence of mutations correlated with high risk FISH (11q- and/or 17p-) and unfavourable cytogenetic (11q-, 17p- or complex karyotype) findings. Patients carrying CLLs with gene mutations showed a significant shorter median time to first treatment in comparison to those without mutations (20 months vs not reached at 76 months). The frequency of mutations in the 20 investigated genes is in line with published data in the literature using whole exome sequencing. This study shows that the simultaneous sequencing of a panel of genes implicated in CLL is feasible. In conclusion, in this work we tried to improve our knowledge on some fundamental pathogenetic aspects of CLL, including: i) the development of genetic lesions in CD38+ activated cells, obtained from the PB in patients in an initial and indolent phase of the disease; ii) the pattern of cytogenetic lesions in lymph node microenvironment (proliferation centres) in patients in a more advanced phase of the disease; iii) to translate this knowledge in clinical practice, we assessed prognosis with modern techniques and we identified cytogenetic lesions associated with disease progression and shorter time to treatment; iv) we identified recurrent genetic lesions potentially useful for further refinement of our ability to predict prognosis and response to treatment

MicroRNAs involvement in fludarabine refractory chronic lymphocytic leukemia

<p>Abstract</p> <p>Background</p> <p>Fludarabine, is one of the most active single agents in the treatment of chronic lymphocytic leukemia (CLL). Over time, however, virtually all CLL patients become fludarabine-refractory. To elucidate whether microRNAs are involved in the development of fludarabine resistance, we analyzed the expression of 723 human miRNAs before and 5-days after fludarabine mono-therapy in 17 CLL patients which were classified as responder or refractory to fludarabine treatment based on NCI criteria.</p> <p>Results</p> <p>By comparing the expression profiles of these two groups of patients, we identified a microRNA signature able to distinguish refractory from sensitive CLLs. The expression of some microRNAs was also able to predict fludarabine resistance of 12 independent CLL patients. Among the identified microRNAs, miR-148a, miR-222 and miR-21 exhibited a significantly higher expression in non-responder patients either before and after fludarabine treatment. After performing messenger RNA expression profile of the same patients, the activation of p53-responsive genes was detected in fludarabine responsive cases only, therefore suggesting a possible mechanism linked to microRNA deregulation in non-responder patients. Importantly, inhibition of miR-21 and miR-222 by anti-miRNA oligonucleotides induced a significant increase in caspase activity in fludarabine-treated p53-mutant MEG-01 cells, suggesting that miR-21 and miR-222 up-regulation may be involved in the establishment of fludarabine resistance.</p> <p>Conclusions</p> <p>This is the first report that reveals the existence of a microRNA profile that differentiate refractory and sensitive CLLs, either before and after fludarabine mono-therapy. A p53 dysfunctional pathway emerged in refractory CLLs and could contribute in explaining the observed miRNA profile. Moreover, this work indicates that specific microRNAs can be used to predict fludarabine resistance and may potentially be used as therapeutic targets, therefore establishing an important starting point for future studies.</p

Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR

Background: Breast cancer circulating biomarkers include carcinoembryonic antigen and carbohydrate antigen 15-3, which are used for patient follow-up. Since sensitivity and specificity are low, novel and more useful biomarkers are needed. The presence of stable circulating microRNAs (miRNAs) in serum or plasma suggested a promising role for these tiny RNAs as cancer biomarkers. To acquire an absolute concentration of circulating miRNAs and reduce the impact of preanalytical and analytical variables, we used the droplet digital PCR (ddPCR) technique. Results: We investigated a panel of five miRNAs in the sera of two independent cohorts of breast cancer patients and disease-free controls. The study showed that miR-148b-3p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than that in controls in both cohorts. For these two miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analyses. In addition, we showed that higher levels of serum miR-10b-5p were associated with clinicobiological markers of poor prognosis. Conclusions: The study revealed the usefulness of the ddPCR approach for the quantification of circulating miRNAs. The use of the ddPCR quantitative approach revealed very good agreement between two independent cohorts in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients versus controls. Overall, this study supports the use of the quantitative ddPCR approach for monitoring the absolute levels of diagnostic and prognostic tumor-specific circulating miRNAs

Sensitive and specific detection of lung cancer DNA in bronchial washing by a novel methylation-specific droplet digital PCR

Rationale: In patients with suspected lung cancer fibrobronchoscopy (FBS) has a key role in the diagnostic process, but its performance is variable (from 90% in the presence of visible lesions to less than 30% for distal ones). DNA methylation analysis emerged as a promising approach for cancer detection. Aberrantly methylated DNA sequences are present in tumour cells and represent cancer specific biomarkers that can be detected in biological fluids. Our study attempts to improve the diagnostic power of BW through the use of cancer specific DNA methylation biomarkers. METHODS: We performed a retrospective case-control study in which we investigated the methylation status of a panel of genes (RASSF1A, CDH1, DLC1 and PRPH) in BWs samples from 91 lung cancer patients and 31 non-cancer controls (current or former smokers). We analysed the methylation status of DNAs from BWs fluids by using a novel highly sensitive two-colour droplet digital methylation-specific PCR. Results: The 4-gene panel exhibited a significant diagnostic power, with a 97% sensitivity and 74% specificity (overall diagnostic accuracy = 0.88), conferring a relative risk of 7.3, at an odds ratio of 76.1 (95% CI from 12.7 to 127). The ROC curve analysis (AUC=0.93) confirmed the excellent diagnostic power of the 4-genes panel. In addition the methylation assay was positive in 35 of the 36 BW samples with negative cytological analyses. CONCLUSIONS. Our results indicate that the aberrant methylation of this panel of genes could represent a helpful support to current diagnostic strategies for lung cancer detection

Absolute quantification of cell-free microRNAs in cancer patients

The hypothesis to use microRNAs (miRNAs) circulating in the blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exciting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post- analytical variables have on the final results. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. Then, we assessed the absolute level of nine different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 207 samples from healthy controls and cancer patients using droplet digital PCR (ddPCR) technology. We identified miRNAs specifically modulated in one or more cancer types, according to tissue source. The significant reduction of miR-181a-5p levels in breast cancer patients serum was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90), with AUC 0.66 and 0.73 respectively. This study finally powers the use of cell-free miRNAs as cancer biomarkers and propose miR-181a-5p as a diagnostic breast cancer biomarker

Characterisation of peripheral blood mononuclear cell microRNA in early onset psoriatic arthritis

OBJECTIVES: To evaluate the micro-RNA (miRNA) expression profile in patients with early psoriatic arthritis (PsA) in order to assess the role of miRNAs as potential PsA biomarkers. METHODS: The expression of 723 mature miRNAs in peripheral blood mononuclear cells of early PsA patients in comparison with early-rheumatoid arthritis (ERA) patients and healthy controls (HC) was evaluated using a miRNA microarray. All patients had active disease and were naïve from treatment. The results were validated for a specific miRNA (miR-21-5p) in the entire series of patients plus an additional group of early PsA, ERA and HC using droplet digital PCR. RESULTS: In PsA, microarray analysis revealed a distinct pattern of 19 (vs. HC) and 48 (vs. ERA) deregulated miRNAs (p<0.05). The significant up-regulation of miR-21-5p both in early PsA and in ERA in comparison with HC was validated and confirmed. In PsA, miR-21-5p was found significantly down regulated after 12 weeks of therapy, which significantly correlated with the reduction of DAPSA score. CONCLUSIONS: In early PsA, a 19- (vs. HC) and 48- (vs. ERA) miRNA signature was identified. A differential expression of miRNAs in patients with active disease makes them attractive biomarkers of psoriatic disease. MiR-21-5p was found up-regulated both in early PsA and ERA, a finding which highlights its role in the inflammatory process in general and its potential role as a therapeutic target in different inflammatory disorders. A potential role of miR-21-5p as a response to treatment biomarker in early PsA has been identified.Objective To evaluate the micro-RNA (miRNA) expression profile in patients with early psoriatic arthritis (PsA) in order to assess the role of miRNAs as potential PsA biomarkers. Methods The expression of 723 mature miRNAs in peripheral blood mononuclear cells of early PsA patients in comparison with early-rheumatoid arthritis (ERA) patients and healthy controls (HC) was evaluated using a miRNA microarray. All patients had active disease and were naïve from treatment. The results were validated for a specific miRNA (miR-21-5p) in the entire series of patients plus an additional group of early PsA, ERA and HC using droplet digital PCR. Results In PsA, microarray analysis revealed a distinct pattern of 19 (vs. HC) and 48 (vs. ERA) deregulated miRNAs (p < 0.05). The significant up-regulation of miR-21-5p both in early PsA and in ERA in comparison with HC was validated and confirmed. In PsA, miR-21-5p was found significantly down regulated after 12 weeks of therapy, which significantly correlated with the reduction of DAPSA score. Conclusion In early PsA, a 19-(vs. HC) and 48-(vs. ERA) miRNA signature was identified. A differential expression of miRNAs in patients with active disease makes them attractive biomarkers of psoriatic disease. MiR-21-5p was found up-regulated both in early PsA and ERA, a finding which highlights its role in the inflammatory process in general and its potential role as a therapeutic target in different inflammatory disorders. A potential role of miR-21-5p as a response to treatment biomarker in early PsA has been identified

In CLL, comorbidities and the complex karyotype are associated with an inferior outcome independently of CLL-IPI

In CLL, comorbidities and the complex karyotype are associated with an inferior outcome independently of CLL-IP

The implementation of a Community Health Centre-based primary care model in Italy. The experience of the Case della Salute in the Emilia-Romagna Region

BACKGROUND: The Comunity Health Centre (CHC) primary care model is a team-based health care delivery model intended to provide comprehensive and continuous medical care to patients within a defined community. The CHC, Case della Salute in Italian, model was introduced in the Emilia-Romagna Region in 2010.METHODS: We present updated data on the implementation on the CHC Case della Salute primary care model in the Emilia-Romagna Region.RESULTS: There are 67 operating CHCs in Emilia-Romagna (update March 2015); 26 small (39%), 24 medium (36%) and 17 large (25%). Since 2011 the number of operating CHCs has increased by 60%, reaching 55% of the target planned CHCs (n. = 122). There is, on average, one running CHC per 66.524 inhabitants. 16% of total general practitioners (GPs) and 8.4% of total family paediatricians working in Emilia-Romagna have their practice in CHCs. CHCs offer primary and specialist integrated care, prevention services, health education and social care.DISCUSSION: Although preliminary results suggest CHCs have fostered primary care's quality and efficiency, more research is needed to assess their impact on improving clinical, social and economic outcomes