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Droplet digital PCR (ddPCR) has been successfully used with TaqMan assays to assess gene expression through the quantification of mRNA and miRNA. Recently, a new ddPCR system that can also run DNA-binding dye-based assays has been developed but it has not yet been tested for miRNA. We tested and compared the feasibility of quantifying miRNA with the new QX200 Droplet Digital PCR system when used with EvaGreen dye– and TaqMan probe–based assays. RNA from plasma and serum of 28 patients with cancer and healthy persons was reverse-transcribed and quantified for two circulating miRNAs and one added exogenous miRNA, with both EvaGreen dye–based miRCURY LNA miRNA assays and TaqMan assays. Amplification and detection of target miRNAs were performed on the QX200 ddPCR system. Conditions required to run miRCURY LNA miRNA assays were optimized. The EvaGreen-based assay was precise, reproducible over a range of concentrations of four orders of magnitude, and sensitive, detecting a target miRNA at levels down to 1 copy/μL. When this assay was compared with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma (Pearson r > 0.90). EvaGreen dye–based and TaqMan probe–based assays can be equally used with the ddPCR system to quantify circulating miRNAs in human plasma and serum. This study establishes the basis for using EvaGreen dye–based assays on a ddPCR system for quantifying circulating miRNA biomarkers and potentially other low-abundance RNA biomarkers in human biofluids.See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.”Cancer Epidemiol Biomarkers Prev; 23(12); 2638–42. ©2014 AACR.

XLSX file - 33K, Features of CLL samples.

Purpose: Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial.Experimental Design: We analyzed the expression of 723 mature miRNAs in 217 early-stage CLL cases and in various different normal B-cell subpopulations from tonsils and peripheral blood.Results: Our analyses indicated that CLL cells exhibited a miRNA expression pattern that was most similar to the subsets of antigen-experienced and marginal zone–like B cells. These normal subpopulations were used as reference to identify differentially expressed miRNAs in comparison with CLL. Differences related to the expression of 25 miRNAs were found to be independent from IGHV mutation status or cytogenetic aberrations. These differences, confirmed in an independent validation set, led to a novel comprehensive description of miRNAs potentially involved in CLL. We also identified miRNAs whose expression was distinctive of cases with mutated versus unmutated IGHV genes or cases with 13q, 11q, and 17p deletions and trisomy 12. Finally, analysis of clinical data in relation to miRNA expression revealed that miR26a, miR532-3p, miR146-5p, and miR29c* were strongly associated with progression-free survival.Conclusion: This study provides novel information on miRNAs expressed by CLL and normal B-cell subtypes, with implication on the cell of origin of CLL. In addition, our findings indicate a number of deregulated miRNAs in CLL, which may play a pathogenic role and promote disease progression. Collectively, this information can be used for developing miRNA-based therapeutic strategies in CLL. Clin Cancer Res; 20(15); 4141–53. ©2014 AACR.

PDF file - 1024K, Supplementary Figure 1. Purification of normal human B cell subset populations. Supplementary Figure 2. CLL miRNA expression profile is homogeneous and distinct from multiple myeloma and Sezary syndrome. Supplementary Figure 3. Quantitative PCR validation of miRNAs differentially expressed between CLL and normal memory B cells. Supplementary Figure 4. Expression of miR-29c* in M-CLL and UM-CLL. Supplementary Figure 5. Validation of some miRNAs differentially expressed in IGHV or FISH classes. Supplementary Figure 6. Cluster analysis of averaged miRNA expression in the different FISH subgroups of CLL.

XLSX file - 248K, microRNAs exhibiting differential expression between CLL and normal antigen-experienced B cells.

Cytopathological analyses of bronchial washings (BWs) collected during fibre‐optic bronchoscopy are often inconclusive for lung cancer diagnosis. To address this issue, we assessed the suitability of conducting molecular analyses on BWs, with the aim to improve the diagnosis and outcome prediction of lung cancer. The methylation status of RASSF1A, CDH1, DLC1 and PRPH was analysed in BW samples from 91 lung cancer patients and 31 controls, using a novel two‐colour droplet digital methylation‐specific PCR (ddMSP) technique. Mutations in ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1 and TP53 and gene fusions of ALK, RET and ROS1 were also investigated, using next‐generation sequencing on 73 lung cancer patients and 14 tumour‐free individuals. Our four‐gene methylation panel had significant diagnostic power, with 97% sensitivity and 74% specificity (relative risk, 7.3; odds ratio, 6.1; 95% confidence interval, 12.7–127). In contrast, gene mutation analysis had a remarkable value for predictive, but not for diagnostic, purposes. Actionable mutations in EGFR, HER2 and ROS1 as well as in other cancer genes (KRAS, PIK3CA and TP53) were detected. Concordance with gene mutations uncovered in tumour biopsies was higher than 90%. In addition, bronchial‐washing analyses permitted complete patient coverage and the detection of additional actionable mutations. In conclusion, BWs are a useful material on which to perform molecular tests based on gene panels: aberrant gene methylation and mutation analyses could be performed as approaches accompanying current diagnostic and predictive assays during the initial workup phase. This study establishes the grounds for further prospective investigation., Diagnostic and predictive assessment remains major challenges in lung cancer management. In this work, we investigate the importance of molecular investigations applied to bronchial washings collected during fibre‐optic bronchoscopy for lung lesions. Using a cancer‐specific DNA methylation panel and gene mutations analyses, we demonstrated that bronchial washing represents a useful material for performing molecular tests in lung cancer patients.

In a series of 349 patients with chronic lymphocytic leukaemia (CLL), we found lower levels of signalling lymphocytic activation molecule family member 1 (SLAMF1) expression in cases with highly complex karyotypes, as defined by the presence of five or more chromosomal abnormalities (CK5; P < 0·001) and with major chromosomal structural abnormalities (P < 0·001). SLAMF1 downregulation was significantly associated with advanced Binet Stage (P = 0·001), CD38 positivity (P < 0·001), high β2 -microglobulin levels (P < 0·001), immunoglobulin heavy chain variable region gene (IGHV) unmutated status (P < 0·001), 11q deletion (P < 0·001), tumour protein p53 (TP53) disruption (P = 0·011) and higher risk CLL International Prognostic Index categories (P < 0·001). Multivariate analysis showed that downregulated SLAMF1 levels had independent negative prognostic impact on time-to-first treatment (P < 0·001) and overall survival (P < 0·001).

The complex karyotype (CK) is an established negative prognostic marker in a number of haematological malignancies. After the introduction of effective mitogens, a growing body of evidence has suggested that the presence of 3 or more aberrations by conventional banding analysis (CBA) is associated with an unfavorable outcome in chronic lymphocytic leukemia (CLL). Thus, the importance of CBA was recognized by the 2018 guidelines of the International Workshop on CLL, which proposed the introduction of CBA in clinical trials to validate the value of karyotype aberrations. Indeed, a number of observational studies showed that cytogenetic aberrations and, particularly, the CK may have a negative independent impact on objective outcome measures (i.e. time to first treatment, progression free survival, time to chemorefractoriness and overall survival) both in patients treated with chemoimmunotherapy and, possibly, in patients receiving novel mechanism-based treatment. Here, we set out to present the scientific evidence supporting the significance of CK as a prognostic marker in CLL and to discuss the biological basis showing that the CK is a consequence of genomic instability.

Clonal evolution of chronic lymphocytic leukemia (CLL) often follows chemotherapy and is associated with adverse outcome, but also occurs in untreated patients, in which case its predictive role is debated. We investigated whether the selection and expansion of CLL clone(s) precede an aggressive disease shift. We found that clonal evolution occurs in all CLL patients, irrespective of the clinical outcome, but is faster during disease progression. In particular, changes in the frequency of nucleotide variants (NVs) in specific CLL-related genes may represent an indicator of poor clinical outcome.

Refined karyotype-based prognostic stratification of chronic lymphocytic leukemia with a low- and very-low-risk genetic profile

We investigated whether karyotype analysis and mutational screening by next generation sequencing could predict outcome in 101 newly diagnosed chronic lymphocytic leukemia patients with high-risk features, as defined by the presence of unmutated IGHV gene and/or 11q22/17p13 deletion by FISH and/or TP53 mutations. Cytogenetic analysis showed favorable findings (normal karyotype and isolated 13q14 deletion) in 30 patients, unfavorable (complex karyotype and/or 17p13/11q22 deletion) in 34 cases and intermediate (all other abnormalities) in 36 cases. A complex karyotype was present in 21 patients. Mutations were detected in 56 cases and were associated with unmutated IGHV status (p = 0.040) and complex karyotype (p = 0.047). TP53 disruption (i.e. TP53 mutations and/or 17p13 deletion by FISH) correlated with the presence of ≥ 2 mutations (p = 0.001) and a complex karyotype (p = 0.012). By multivariate analysis, an advanced Binet stage (p < 0.001) and an unfavorable karyotype (p = 0.001) predicted a shorter time to first treatment. TP53 disruption (p = 0.019) and the unfavorable karyotype (p = 0.028) predicted a worse overall survival. A shorter time to chemorefractoriness was associated with TP53 disruption (p = 0.001) and unfavorable karyotype (p = 0.025). Patients with both unfavorable karyotype and TP53 disruption presented a dismal outcome (median overall survival and time to chemorefractoriness of 28.7 and 15.0 months, respectively). In conclusion, karyotype analysis refines risk stratification in high-risk CLL patients and could identify a subset of patients with highly unfavorable outcome requiring alternative treatments.

In the Local Health Authority (LHA) of Parma, Emilia Romagna, Italy, 16 medical homes were established between 2011 and 2014. The authors implemented a 1-year (January 1, 2015, to December 31, 2015) cross-sectional population-based design to compare utilization and processes of care between medical homes and comparison practices using the Parma LHA administrative health care database. Residents (n = 372 396) attributed to a primary care physician practicing in a medical home as of January 1, 2015, were considered exposed to medical homes. Adjusted rates of emergency department (ED) use (incidence rate ratio [IRR] = 0.86; 95% CI = 0.82-0.90), potentially avoidable ED use (IRR = 0.78; 95% CI = 0.72-0.84), and hospitalization for chronic ambulatory care sensitive conditions (ACSCs; IRR = 0.87, 95% CI = 0.78-0.97) were lower among patients in medical homes. Performance on process of care measures favored the medical home group; however, associations were generally weak. Receipt of care in medical homes in Parma LHA was associated with lower rates of avoidable ED visits and hospitalizations for chronic ACSCs.

Aim: To investigate the genome-wide methylation of genetically characterized colorectal cancer stem cell (CR-CSC) lines. Materials & methods: Eight CR-CSC lines were isolated from primary colorectal cancer (CRC) tissues, cultured and characterized for aneuploidy, mutational status of CRC-related genes and microsatellite instability (MSI). Genome-wide DNA methylation was assessed by MethylationEPIC microarray. Results: We describe a distinctive methylation pattern that is maintained following in vivo passages in immune-compromised mice. We identified an epigenetic CR-CSC signature associated with MSI. We noticed that the preponderance of the differentially methylated positions do not reside at CpG islands, but spread to shelf and open sea regions. Conclusion: Given that CRCs with MSI-high status have a lower metastatic potential, the identification of a MSI-related methylation signature could provide new insights and possible targets into metastatic CRC.

To clarify whether karyotype aberrations (KA) involving regions not covered by the standard fluorescence in situ hybridization (FISH) panel have independent prognostic relevance, we evaluated KA by conventional cytogenetics in a learning cohort (LC; n = 166) and a validation cohort (VC; n = 250) of untreated chronic lymphocytic leukemia (CLL) patients. In the VC, novel mitogens were used to improve metaphase generation and TP53, NOTCH1, and SF3B1 mutations were assessed. KA undetected by FISH were found in 35 and 35% of the cases in the LC and VC, respectively. In addition to FISH, KA allowed reclassification of 23 and 26% of cases in the LC and VC, respectively, into a higher cytogenetic risk group. By multivariate analysis, both in the LC and VC, KA other than isolated 13q deletion correlated with a shorter time to first treatment (TFT; P < 0.001 and 0.003, respectively), while a complex karyotype predicted a worse overall survival (OS, P = 0.015 and 0.010, respectively). In the VC, where a comprehensive biologic assessment was performed, a shorter TFT was also predicted by stage (P < 0.001), IGHV mutational status (P = 0.05), and del(17p)/TP53 mutations (P = 0.033) while stage (P = 0.023) and del(17p)/TP53 mutations (P = 0.024) independently predicted a shorter OS. FISH results did not independently impact on TFT and OS, in the LC and VC cohorts; this was also the case for NOTCH1 and SF3B1 mutations in the VC. We suggest that in CLL, conventional karyotyping with novel mitogens could be more effective than FISH for the detection of KA allowing for a more precise refinement of prognosis.

Complex karyotype (CK) is a negative prognostic factor in chronic lymphocytic leukaemia (CLL). However, CK is a heterogeneous cytogenetic category. Unbalanced rearrangements were present in 73·3% of 90 CLL patients with CK (i.e. ≥3 chromosome aberrations in the same clone), and were associated with a shorter overall survival (P = 0·025) and a shorter time to first treatment (P = 0·043) by multivariate analysis. Patients with unbalanced rearrangements presented a distinct mRNA expression profile. In conclusion, CLL patients with unbalanced rearrangements might represent a subset of very high-risk CLL patients with distinct clinical and biological characteristics.

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.

To the editor: Chronic lymphocytic leukemia (CLL) represents the most common form of leukemia in Western countries.[1][1] The clinical course of the disease is quite heterogeneous with some patients living for years with asymptomatic disease and others experiencing early progression and requiring

Purpose: Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial. Experimental Design: We analyzed the expression of 723 mature miRNAs in 217 early-stage CLL cases and in various different normal B-cell subpopulations from tonsils and peripheral blood. Results: Our analyses indicated that CLL cells exhibited a miRNA expression pattern that was most similar to the subsets of antigen-experienced and marginal zone–like B cells. These normal subpopulations were used as reference to identify differentially expressed miRNAs in comparison with CLL. Differences related to the expression of 25 miRNAs were found to be independent from IGHV mutation status or cytogenetic aberrations. These differences, confirmed in an independent validation set, led to a novel comprehensive description of miRNAs potentially involved in CLL. We also identified miRNAs whose expression was distinctive of cases with mutated versus unmutated IGHV genes or cases with 13q, 11q, and 17p deletions and trisomy 12. Finally, analysis of clinical data in relation to miRNA expression revealed that miR26a, miR532-3p, miR146-5p, and miR29c* were strongly associated with progression-free survival. Conclusion: This study provides novel information on miRNAs expressed by CLL and normal B-cell subtypes, with implication on the cell of origin of CLL. In addition, our findings indicate a number of deregulated miRNAs in CLL, which may play a pathogenic role and promote disease progression. Collectively, this information can be used for developing miRNA-based therapeutic strategies in CLL. Clin Cancer Res; 20(15); 4141–53. ©2014 AACR.

Treatment of chronic lymphocytic leukemia (CLL) has dramatically changed over the last years, with significant improvement in overall survival (OS) and increased efficacy in genetically defined "high-risk" disease. Besides prospective clinical trials usually enrolling young and fit patients, retrospective studies were performed comparing the outcome of patients belonging to different age groups and showing longer survival in patients diagnosed in the most recent periods. In patients younger than 70 years the 10-year relative survival was 43-53% in the 1980s as compared with 59-63% in the 2000s. Likewise, the 10-year relative survival in patients >70 years was 22-42% in the 1980s and 46-55% in the 2000s. Improved outcome derived in part by the introduction of effective regimens in genetically defined "high-risk" disease (i.e., 17p-, 11q-, TP53, NOTCH1, SF3B1 mutations), especially in the younger and/or fit patients. The unfavorable prognostic significance of 11q- was overcome by chemoimmunotherapy. High-dose steroids with anti-CD52 appeared to improve the response rate in 17p-/TP53 mutated cases and allogeneic transplantation achieved prolonged disease control irrespective of high-risk disease. Further improvement is being generated by the new anti-CD20 obinutuzumab in the elderly and by mechanism-based treatment using kinase-targeting agents or anti-BCL2 molecules yielding high-response rate and impressive progression-free survival in the chemorefractory setting as well as in previously untreated patients.

Dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). The Eμ-TCL1 transgenic mouse develops a form of leukemia that is similar to the aggressive type of human B-CLL, and this valuable model has been widely used for testing novel therapeutic approaches. Here, we adopted this model to investigate the potential effects of miR-26a, miR-130an and antimiR-155 in CLL therapy. Improved delivery of miRNA molecules into CLL cells was obtained by developing a novel system based on lipid nanoparticles conjugated with an anti-CD38 monoclonal antibody. This methodology has proven to be highly effective in delivering miRNA molecules into leukemic cells. Short- and long-term experiments showed that miR-26a, miR-130a and anti-miR-155 increased apoptosis after in vitro and in vivo treatment. Of this miRNA panel, miR-26a was the most effective in reducing leukemic cell expansion. Following long-term treatment, apoptosis was readily detectable by analyzing cleavage of PARP and caspase-7. These effects could be directly attributed to miR-26a, as confirmed by significant downregulation of its proven targets, namely cyclin-dependent kinase 6 and Mcl1. The results of this study are relevant to two distinct areas. The first is related to the design of a technical strategy and to the selection of CD38 as a molecular target on CLL cells, both consenting efficient and specific intracellular transfer of miRNA. The original scientific finding inferred from the above approach is that miR-26a can elicit in vivo anti-leukemic activities mediated by increased apoptosis.

Chronic lymphocytic leukemia (CLL) displays an extremely variable clinical behaviour. Accurate prognostication and prediction of response to treatment is important in an era of effective first-line regimens and novel molecules for high risk patients. Because a plethora of prognostic biomarkers were identified, but few of them were validated by multivariable analysis in comprehensive prospective studies, we applied in this survey stringent criteria to select papers from the literature in order to identify the most reproducible prognostic/predictive markers. Each biomarker was analysed in terms of reproducibility across the different studies with respect to its impact on time to first treatment (TTFT), progression free survival (PFS), overall survival (OS), response to treatment and transformation into Richter’s syndrome (RS). We were able to identify the following biomarkers as the most reliable in guiding risk stratification in the daily clinical practice: 17p-/TP53 mutations, IGHV unmutated configuration, short telomeres and 11q-. However, the method for measuring telomere length was not validated yet and 11q- was predictive of inferior OS only in those patients who did not receive FCR-like combinations. Stage and lymphocytosis were predictive of shorter TTFT and age, high serum thymidine kinase levels and poor performance status were predictive of shorter OS. Using our stringent criteria no parameter was found to independently predict for inferior response to treatment or development of RS.

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.We present updated data on the implementation on the CHC Case della Salute primary care model in the Emilia-Romagna Region.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.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.

Background In chronic lymphocytic leukemia (CLL), next-generation sequencing (NGS) analysis represents a sensitive, reproducible, and resource-efficient technique for routine screening of gene mutations. Methods We performed an extensive biologic characterization of newly diagnosed CLL, including NGS analysis of 20 genes frequently mutated in CLL and karyotype analysis to assess whether NGS and karyotype results could be of clinical relevance in the refinement of prognosis and assessment of risk of progression. The genomic DNA from peripheral blood samples of 200 consecutive CLL patients was analyzed using Ion Torrent Personal Genome Machine, a NGS platform that uses semiconductor sequencing technology. Karyotype analysis was performed using efficient mitogens. Results Mutations were detected in 42.0 % of cases with 42.8 % of mutated patients presenting 2 or more mutations. The presence of mutations by NGS was associated with unmutated IGHV gene (p = 0.009), CD38 positivity (p = 0.010), risk stratification by fluorescence in situ hybridization (FISH) (p

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. 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.

Droplet digital PCR (ddPCR) has been successfully used with TaqMan assays to assess gene expression through the quantification of mRNA and miRNA. Recently, a new ddPCR system that can also run DNA-binding dye-based assays has been developed but it has not yet been tested for miRNA. We tested and compared the feasibility of quantifying miRNA with the new QX200 Droplet Digital PCR system when used with EvaGreen dye– and TaqMan probe–based assays. RNA from plasma and serum of 28 patients with cancer and healthy persons was reverse-transcribed and quantified for two circulating miRNAs and one added exogenous miRNA, with both EvaGreen dye–based miRCURY LNA miRNA assays and TaqMan assays. Amplification and detection of target miRNAs were performed on the QX200 ddPCR system. Conditions required to run miRCURY LNA miRNA assays were optimized. The EvaGreen-based assay was precise, reproducible over a range of concentrations of four orders of magnitude, and sensitive, detecting a target miRNA at levels down to 1 copy/μL. When this assay was compared with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma (Pearson r > 0.90). EvaGreen dye–based and TaqMan probe–based assays can be equally used with the ddPCR system to quantify circulating miRNAs in human plasma and serum. This study establishes the basis for using EvaGreen dye–based assays on a ddPCR system for quantifying circulating miRNA biomarkers and potentially other low-abundance RNA biomarkers in human biofluids. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2638–42. ©2014 AACR.

// Gian Matteo Rigolin 1 , Elena Saccenti 1,2 , Lara Rizzotto 1 , Manuela Ferracin 2 , Sara Martinelli 1 , Luca Formigaro 1 , Francesca Cibien 1 , Maurizio Cavallari 1 , Enrico Lista 1 , Giulia Daghia 1 , Olga Sofritti 1 , Maria Ciccone 1 , Francesco Cavazzini 1 , Laura Lupini 2 , Cristian Bassi 2 , Barbara Zagatti 2 , Massimo Negrini 2 , Antonio Cuneo 1 1 Hematology Section, Department of Medical Sciences, University of Ferrara, University Hospital Arcispedale S. Anna, Ferrara, Italy 2 Laboratory for Technologies of Advanced Therapies (LTTA) and Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy Correspondence: Gian Matteo Rigolin, email: // Keywords : Oncotargets, miR-125a-5p, CLL, CD38, prognosis Received : September 8, 2013 Accepted : October 28, 2013 Published : October 30, 2013 Abstract The majority of patients with chronic lymphocytic leukemia (CLL) and favorable prognostic features live for long periods without treatment. However, unexpected disease progression is observed in some cases. In a cohort of untreated CD38- CLL patients with normal FISH or isolated 13q- we found that, by fluorescence in situ hybridization (FISH), 16/28 cases presented, within immunomagnetic sorted CD38+ cells, genetic lesions undetectable in the CD38- fraction. These patients showed a shorter time to first treatment (TTFT, p=0.0162) in comparison to cases without FISH lesions in CD38+ cells. Patients with FISH abnormalities in CD38+ cells showed a distinctive microRNA profile, characterized by the down-regulation of miR-125a-5p both in the CD38- and CD38+ populations. In an independent cohort of 71 consecutive untreated CD38- CLL with normal FISH or isolated 13q-, a lower miR125a-5p expression was associated with a shorter TTFT both in univariate and multivariate analysis (p=0.003 and 0.016, respectively) and with a higher prevalence of mutations (7/12 vs 0/8, p=0.015) as assessed by next-generation sequencing. In conclusion, our data showed previously unrecognized subclonal heterogeneity within the CD38+ fraction of CD38- CLL patients with low-risk FISH findings and suggested an association between down-regulated miR-125a-5p expression, genetic complexity and worse outcome.

It is unclear whether karyotype aberrations that occur in regions uncovered by the standard fluorescence in situ hybridization (FISH) panel have prognostic relevance in chronic lymphocytic leukemia (CLL). We evaluated the significance of karyotypic aberrations in a learning cohort (LC; n = 64) and a validation cohort (VC; n = 84) of patients with chronic lymphocytic leukemia with “normal” FISH. An abnormal karyotype was found in 21.5% and 35.7% of cases in the LC and VC, respectively, and was associated with a lower immunophenotypic score (P = .030 in the LC, P = .035 in the VC), advanced stage (P = .040 in the VC), and need for treatment (P = .002 in the LC, P = < .0001 in the VC). The abnormal karyotype correlated with shorter time to first treatment and shorter survival in both the LC and the VC, representing the strongest prognostic parameter. In patients with chronic lymphocytic leukemia with normal FISH, karyotypic aberrations by conventional cytogenetics with novel mitogens identify a subset of cases with adverse prognostic features.

To better define the significance of clonal evolution (CE) including 14q32 translocations involving the immunoglobulin heavy chain gene (IGH) in chronic lymphocytic leukemia (CLL), 105 patients were analyzed sequentially by fluorescence in situ hybridization (FISH) with the following panel of probes: 13q14/D13S25, 11q22/ATM, 17p13/TP53, #12-centromere and 14q32/IGH break-apart probe. CE was observed in 15/105 patients after 24-170 months (median 64). Recurring aberrations at CE were 14q32/IGH translocation in seven patients; other aberrations were 17p -, 11q -, biallelic 13q - and 14q32 deletion. CE was detected in 15/58 pre-treated patients; in contrast, none of 47 untreated patients developed CE (p0.0001). In two cases the appearance of 14q32/IGH translocation was first detected in the bone marrow (BM) or in the lymph node (LN) and 13-58 months later in the peripheral blood (PB). ZAP70 + and high-risk cytogenetics predicted for the occurrence of CE with borderline statistical significance (p = 0.055 and 0.07, respectively). Shorter time to first treatment (TTT) and time to chemorefractoriness (TTCR) were noted in 15 patients with CE when compared to patients without CE (TTT: 35 vs. 71 months, p = 0.0033 and TTCR: 34 vs. 86 months, p = 0.0046, respectively). Survival after the development of CE was 32 months (standard error 8.5). We arrived at the following conclusions: (i) 14q32/IGH translocation may represent one of the most frequent aberrations acquired during the natural history of CLL and (ii) it may be detected earlier in BM or LN samples; (iii) CE including 14q32/IGH translocation occurs in pre-treated patients with short TTT and TTCR; (iii) survival after CE is relatively short.

To better define the significance of proliferation centers (PCs), the morphological hallmark of chronic lymphocytic leukemia (CLL), lymph node biopsies taken from 183 patients were submitted to histopathologic and fluorescence in situ hybridization (FISH) studies using a 5-probe panel on tissue microarrays. Seventy-five cases (40.9%) with confluent PCs were classified as 'PCs-rich' and 108 cases (59.1%) with scattered PCs were classified as 'typical'. Complete FISH data were obtained in 101 cases (55.1%), 79 of which (78.2%) displayed at least one chromosomal aberration. The incidence of each aberration was: 13q- 36,7%, 14q32 translocations 30.8%, 11q- 24.7%, trisomy 12 19.5% and 17p- 15.6%. Five cases showed extra copies of the 14q32 region. The 'PCs-rich' group was associated with 17p-, 14q32/IgH translocation, +12, Ki-67>30%. The median survival from the time of tissue biopsy for PCs-rich and typical groups was 11 and 64 months, respectively (P=0.00001). The PCs-rich pattern was the only predictive factor of an inferior survival at multivariate analysis (P=0.022). These findings establish an association between cytogenetic profile and the amount of PC in CLL, and show that this histopathologic characteristic is of value for risk assessment in patients with clinically significant adenopathy.

Acute leukaemia is a neoplastic disorder of haematopoietic precursors characterized by a rapid clinical course and a relatively high early death rate in spite of recent therapeutic progress. Prompt and reliable diagnostic and prognostic assessment has a favourable impact on patient outcome. Diagnostic suspicion relies on signs and symptoms of bone marrow failure or quali-/quantitative abnormalities of blood cells, which are accurately detected by modern automated counters. Cytomorphological examination of a stained blood and bone marrow film plays a key role in the diagnostic process, with relevant additional information coming from immunophenotyping of blast cells. Cytogenetic and molecular genetic data are the basis of prognostic stratification. In this context, a coordinated intervention of specialized laboratories combining solid expertise in each field involved in the diagnostic work-up is essential; at the same time, the availability of all the required technologies at the same hospital structure would probably lack efficiency due to the low number of tests performed. The organization of laboratory networks, either at regional or national level, especially in the context of clinical trials, may offer a great opportunity for centralization of more sophisticated technologies in reference laboratories, each highly specialized in a particular field of investigation and all interconnected. The challenge for laboratory haematology is the reorganization of clinical and scientific activity according to this model, without loosing educational potential in favour of new generations of medical doctors, haematologists, biologists and laboratory technicians.

Abstract 1459 Background. Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. Adverse prognostic parameters include stage, CD38 and/or ZAP70 expression, the unmutated configuration of the variable region of the Ig heavy chain gene (IGHV) and the presence of specific chromosome aberrations. Using fluorescence in situ hybridization (FISH) with probes detecting trisomy 12 and deletions at 13q14, 11q22–23/ATM and 17p13/p53, approximately 60–80% of the cases carry an abnormality. Patients without FISH aberrations (“normal” FISH) have a relatively favourable outcome. Improved mitotic stimulation using oligonucleotide (odn) and interleukin-2 (IL-2) can detect karyotype aberrations in chromosome regions not covered by the classical 4-probe panel. This study was designed to assess whether the presence of karyotypic aberrations in CLL patients with “normal” FISH may have a correlation with established clinical and prognostic parameters. Methods. Eighty-four patients with “normal” FISH results referred to our laboratory between 2007 and June 2011 were included in the present report. These patients represent consecutive CLL cases diagnosed and treated, according to the NCI criteria, at 4 GIMEMA CLL group centres between 2006 and 2011. Indications for treatment included advanced stage (Binet C) or disease progression. The Matutes immunophenotypic score was calculated giving 1 point to CD5+, CD23+, CD22weak+, sIg weak+ and FMC7- and only patients with a score ≥3 (i.e. typical CLL) were included. The coexpression of the CD38 and CD19 antigens and ZAP-70 was tested on fresh PB cells with a 20% cut-off for positivity. IGVH genes were amplified from genomic DNA and sequenced according to standard methods and the cut-off of 98% homology to the germline sequence was chosen to discriminate between mutated ( Results. An abnormal karyotype was found in 30/84 cases (35,7%) with “normal” FISH. The chromosome aberrations affected regions not covered by the 4-probe panel used by FISH. Recurring aberrations were: interstitial deletions of 14q and of 7q in 5 and 3 cases, respectively; 14q32 translocations in 2 cases, balanced and unbalanced structural changes in 1 case each. A complex karyotype with 3 or more aberrations was found in 12 patients. The correlations between karyotype and clinicobiological parameters are shown in tables 1 and 2.Table 1:Correlation between karyotype and salient clinicobiologic parametersParameterNormal karyotype (54 cases)Abnormal karyotype (30 cases)p (Fischer's exact test)Binet Stage A/B/C45/9/020/7/30.04Matutes score3/4/54/24/257/6/120.035CD38pos/neg11/4112/180.079ZAP-70 pos/neg16/3115/110.083IGHV Unmutated/ Mutated4/208/80.037Table 2:Factors affectingTTFT in univariate snalysisNumber of casesMedian TTFT months (se)pKaryotype Normal Abnormal50 34Not reached at 54 months 18 months (6.7) At multivariate analysis, the following factors were independently predictive of shorter TTFT: advanced disease Binet stage (p=0.049, Hazard Ratio 2.39 [se: 1.06]), and abnormal karyotype (p Conclusions. Conventional karyotyping using odn + IL-2 as mitogens is an effective method for the detection of chromosome aberrations in approximately 1/3 of patients with “normal” FISH on a conventional 4-probe panel. Recurrent aberrations include interstitial deletions at 14q and 7q. The current data show that, in CLL patients with “normal” FISH, conventional cytogenetic analysis using odn + IL-2 identifies a subset of cases with adverse clinical and prognostic features. Disclosures: Rizzotto: CELGENE CORPORATION: Research Funding. Cuneo:Roche: Consultancy, Speakers Bureau.

Background 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. Results 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. Conclusions 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.

Background Micro-RNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression.It is known that an altered miRNA expression plays an important role in cancer.A new emerging role for miRNAs has been evidenced in the pathogenesis of rheumatic diseases such as rheumatoid arthritis and psoriasis.Recently,we evaluated miRNA expression in peripheral blood mononuclear cells (PBMCs) of 21 patients with psoriatic arthritis (PsA) of recent onset and naive from treatment by miRNA microarray. Three mi-RNAs (miR-21-5p, miR-34a and miR-21-3p) resulted significantly upregulated compared to 12 healthy controls. Objectives We set out to validate microarray results in the same series of PsA patients and controls;and to check for changes in the mi-RNA expression from baseline after appropriate therapy. Methods We performed a quantitative RT-PCR (RT-qPCR) analysis for miR-21 in the entire series of 21 PsA patients and 12 controls. We also checked for changes in miR-21 expression profile from baseline after 3 months of appropriate therapy (symptomatic therapy, DMARDs,TNF-alpha blockers) in 9 of the 21 PsA patients. Mature miRNA expression was evaluated by Taqman miRNA assays (Applied Biosystem) specific for miR-21 and RNU6B as reference gene, according to the manufacturer9s protocol. Five ng of total RNA was reverse transcribed using the specific looped primer; quantitative PCR was conducted using the standard Taqman MiRNA assay. Each sample was analyzed in triplicate.The amount of target, normalized on U6 RNA amount, was calculated using 2-ΔCt (Comparative Ct) method. Significance in RT-qPCR results was determined by unpaired t test with Welch9s correction and graphs were created using GraphPad software Results In comparison with healthy controls, the up-regulation of miR-21 in PsA was validated and confirmed by RT-qPCR (p Conclusions The upregulation of miR-21 in early, active and naive of treatment PsA was confirmed and validated. Compared to baseline, miR-21 resulted significantly reduced after 3 months of appropriate therapy. These results, if confirmed on a larger number of patients, could have two important implications. First, it calls into question all the previous studied on miRNA performed on PBMC of pharmacologically treated patients. Second, it opens new horizons not only in understanding pathogenic mechanisms of the disease but also in identifying useful diagnostic and prognostic biomarkers and new possible therapeutic targets in PsA. For the latter purpose, comparative studies with other chronic inflammatory conditions other than PsA are needed References Ciancio G,Ferracin E, Zagatti B,et al. Microrna Expression Profiles in Peripheral Blood Mononuclear Cells of Early Onset Psoriatic Arthritis. Arthritis Rheumatism, 2012, 64(10 Suppl):433. Alevizos I, Illei GG: MicroRNAs as biomarkers in rheumatic diseases. Nat Rev Rheumatol 2010, 6:391-398 Acknowledgements The authors gratefully acknowledge the support of the Regione Emilia Romagna and the University of Ferrara which made this study possible. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4448

Background Rheumatoid arthritis(RA) is a chronic inflammatory joint disorder characterized by immune-driven inflammation of synovial membrane which results in erosion, joint destruction and disability. The etiology of RA is still unknown and many uncertainties regarding its pathogenetic mechanisms persist. Micro-RNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression. Emerging evidence has linked mi-RNA altered expression with pathogenic mechanisms of neoplastic and more recently autoimmune and rheumatic diseases, including RA. Moreover, in the field of rheumatic diseases, miRNAs are today a new exciting area of research as potential biomarkers. Many recent studies have demonstrated that different miRNAs are significantly disregulated (up- or down-regulated) in peripheral blood mononuclear cells (PBMC) and/or synovial cells from RA when compared with healthy controls or osteoarthritis (OA). Of particular interest are today considered miR-155 and miR-146a, which have been found up-regulated both in RA synovial tissue and PBMC, and miR-223, which has been found up-regulated in RA T-lymphocytes from peripheral blood. Objectives Our purpose was to verify whether specific miRNAs are differentially modulated in very early RA in comparison with healthy controls. Methods Blood samples from 10 consecutive patients with early and naive from any treatment RA (disease duration in months: 2±1,4; M:5; F:5; mean age: 57,8±0,48 ) were collected. All patients fulfilled the 2010 ACR/EULAR criteria for RA classification and had active disease (DAS28: 5,34 ± 0,48). 8/10 patients were rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibodies positive. As controls, 12 random healthy volunteers (M:4; W:8; mean age 34 ± 11) were recruited. The expression levels of 723 mature miRNAs in peripheral blood mononuclear cells (PMBC) were investigated in all patients and controls by using Agilent miRNA microarray. Hybridization and data analysis was performed as already described. Differentially expressed genes were identified by using GeneSpring GX software (Agilent Technologies) and moderated t-test with p Results We identified 23 microRNAs significantly upregulated in RA patients ( p ) compared to healthy controls. Among these, miR-21-3p appeared of special interest because of the extent of its modulation and its biological significance. MiR-146a, miR-155 and miR-223 resulted not up-regulated in PBMCs of our early-RA patients in comparison with controls. Conclusions A 23-miRNA signature was identified in patients with early active RA. These data seem to confirm that some miRNAs could have important implications in reflecting the underlying pathogenic processes of RA. Moreover, their expression in patients with active and early disease makes them attractive as potential biomarkers of disease. However, more studies on larger series in patients naive from treatmentare needed in order to identify miRNA specifically correlated to RA. Disclosure of Interest None Declared

Abstract 2429 Up to 80% of Chronic Lymphocytic Leukemia (CLL) harbour clonal chromosome aberrations having important clinical implications (i.e. 13q deletion, +12, 11q/ATM and 17p/TP53 deletions). 14q32/IGH rearrangements were recently found in 6–19% of CLL patients and were associated with therapy-demanding disease and inferior outcome. Whereas evidence was provided that some of the classical aberrations, such as 11q-, 17p-, may appear late in CLL clinical history, no information is presently available concerning 14q32/IGH translocations. The aim of this study was i) to analyze the incidence of 14q32/IGH translocations occurring at clonal evolution in CLL, ii) to analyze the clinicobiologic significance of late-appearing 14q32/IGH translocations. One hundred-five CLL cases seen at our institution in a 10-year period were submitted to FISH analysis at diagnosis or before 1st line treatment as part of routine diagnostic workup. In 47 patients with indolent disease (untreated or treated with 1 line without relapse, group 1) FISH analysis was repeated after 48–96 months (median 72). In 58 relapsed patients who started 2nd line treatment (group 2), FISH was performed sequentially before administration of the 2nd line and before each subsequent line of therapy. These 105 patients fulfilled the following criteria: a) diagnosis of bona fide CLL based on morphology and immunophenotyping (CD5/CD19+, CD23+ as minimal requirement), b) clinical records available for review, c) successful FISH analysis at diagnosis and during follow-up. Those cases with t (11;14)(q13;q32)/CCND1-IGH or other 14q32/IGH translocations present at diagnosis were excluded from this study. Sequential FISH studies were performed in all patients on peripheral blood (PB) samples using commercially available probes for the identification of deletions at 13q14, 11q22/ATM, 17p13/TP53, of trisomy 12 and of 14q32/IGH translocations. In 10 patients bone marrow (BM) aspiration and/or lymph node (LN) biopsy were studied by FISH as well. The patients were treated at disease progression as defined by NCI criteria. Refractory disease was defined by stable disease or progressive disease during treatment or disease progression within 6 months of from antileukemic treatment using fludarabine alone or in combination with other agents. Time to chemorefractoriness was measured from date of first line treatment to date of refractoriness to fludarabine containing regimen or date of last follow-up. Overall survival was measured from diagnosis to date of last follow-up or death and from initiation of first line treatment to the date of death or last follow-up. At diagnosis 39% of the cases had 13q-, 14% had +12, 7% had 11q- and 3% had 17p-. A late-appearing 14q32/IGH rearrangement was not detected among 47 patients in group 1, whereas 7/58 cases (12,1%) in group 2 showed a 14q32/IGH break in 16–25% of the cells. These 7 patients had the following aberrations at diagnosis: 13q- and 11q- in 1 case, 13q- in 2 cases; 11q- in 1 case, +12 in 2 cases, no aberrations 1 case. The 14q32 translocation appeared after a median time of 64 months (range 51–91). It was associated with the appearance of 17p- in 3/7 cases with one of these presenting also biallelic del13q. In two cases paired BM or LN sample and PB samples were available for FISH studies and the appearance of IgH translocation in the BM or in the LN sample preceded its appearance in PB by 13–58 months. All 7 cases with late appearing 14q32/IGH translocation developed chemorefractoriness to fludarabine regimen with a median TTC of 27 months (range 12–40 months), as compared with a TTC of 67 months (range 1–143 months) in 51 treated patients who did not develop the 14q32 translocation (p=0.0002). Overall survival did not differ significantly either when measured from diagnosis or from 1st line treatment in 7 patients with 14q32 translocation as compared with the appropriate control. We arrived at the following conclusions: i) a late-appearing 14q32/IGH translocation occurred at a relatively high incidence (12,1%) in patients with relapsing disease and not in patients with stable disease, ii) this aberration involved a minority of cells and, in approximately half of the cases, it was associated with other aberrations, reflecting complex clonal evolution, iii) in 2 assessable cases it first appeared first in the BM or LN; iv) the appearance of 14q32/IGH translocation was associated with shorter TTC. Disclosures: No relevant conflicts of interest to declare.

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. Electronic supplementary material The online version of this article (doi:10.1186/s40364-015-0037-0) contains supplementary material, which is available to authorized users.

Background In chronic lymphocytic leukemia (CLL), next-generation sequencing (NGS) analysis represents a sensitive, reproducible, and resource-efficient technique for routine screening of gene mutations. Methods We performed an extensive biologic characterization of newly diagnosed CLL, including NGS analysis of 20 genes frequently mutated in CLL and karyotype analysis to assess whether NGS and karyotype results could be of clinical relevance in the refinement of prognosis and assessment of risk of progression. The genomic DNA from peripheral blood samples of 200 consecutive CLL patients was analyzed using Ion Torrent Personal Genome Machine, a NGS platform that uses semiconductor sequencing technology. Karyotype analysis was performed using efficient mitogens. Results Mutations were detected in 42.0 % of cases with 42.8 % of mutated patients presenting 2 or more mutations. The presence of mutations by NGS was associated with unmutated IGHV gene (p = 0.009), CD38 positivity (p = 0.010), risk stratification by fluorescence in situ hybridization (FISH) (p