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Acute promyelocytic leukemia (APL) patients carry in 27% of cases an activating mutation of the fms-like tyrosine kinase-3 (FLT3) gene: internal tandem duplication (ITD) or tyrosine kinase domain (TKD) point mutation. The simultaneous presence of both types of mutations, so-called FLT3 dual mutations, has been reported in 2% of APL, but this circumstance has never been studied. We studied a cohort of 74 APL cases, performing an in-depth analysis of three FLT3 dual mutant cases. Nanopore sequencing (NS) allowed us to characterize their complex mutational profile, showing the occurrence of multiple activating FLT3 mutations on different alleles in the leukemic promyelocytes and suggesting a cumulative impact of these events on the constitutive activation of the FLT3 pathway in APL cells. NS approach not only sheds light on the FLT3 mutational complexity in APL, but may also be useful to better clarify the FLT3 mutations landscape in acute myeloid leukemia.

Interstitial deletions of the long arm of chromosome 12 are rare, with a dozen patients carrying a deletion in 12q21 being reported. Recently a critical region (CR) has been delimited and could be responsible for the more commonly described clinical features, such as developmental delay/intellectual disability, congenital genitourinary and brain malformations. Other, less frequent, clinical signs do not seem to be correlated to the proposed CR. We present seven new patients harboring non-recurrent deletions ranging from 1 to 18.5 Mb differentially scattered across 12q21. Alongside more common clinical signs, some patients have rarer features such as heart defects, hearing loss, hypotonia and dysmorphisms. The correlation of haploinsufficiency of genes outside the CR to specific signs contributes to our knowledge of the effect of the deletion of this gene-poor region of chromosome 12q. This work underlines the still important role of copy number variations in the diagnostic setting of syndromic patients and the positive reflection on management and family genetic counseling.

The evaluation of the somatic hypermutation of the clonotypic immunoglobulin heavy variable gene has become essential in the therapeutic management in chronic lymphocytic leukemia patients. European Research Initiative on Chronic Lymphocytic Leukemia promotes good practices and standardized approaches to this assay but often they are labor-intensive, technically complex, with limited in scalability. The use of next-generation sequencing in this analysis has been widely tested, showing comparable accuracy and distinct advantages. However, the adoption of the next generation sequencing requires a high sample number (run batching) to be economically convenient, which could lead to a longer turnaround time. Here we present data from nanopore sequencing for the somatic hypermutation evaluation compared to the standard method. Our results show that nanopore sequencing is suitable for immunoglobulin heavy variable gene mutational analysis in terms of sensitivity, accuracy, simplicity of analysis and is less time-consuming. Moreover, our work showed that the development of an appropriate data analysis pipeline could lower the nanopore sequencing error rate attitude.

Acute promyelocytic leukemia (APL) patients carry in 27% of cases an activating mutation of the fms-like tyrosine kinase-3 (

We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.

The breakpoint cluster region-abelson 1 p190 fusion transcript is the most frequent variant observed in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). Qualitative-PCR and real-time quantitative PCR are the currently used methods to monitor minimal residual disease (MRD) in Ph+ ALL patients; for the latter, full standardization and an international quality validation are lacking. Here, we developed a droplet digital PCR (ddPCR) assay for MRD monitoring in p190+ ALL cases. The analytical performance was assessed by the limit-of-detection determination, showing a reliability, sensitivity, and precision of the assay of up to 0.001%. Comparison of results obtained with qualitative PCR and ddPCR in 117 follow-up samples from 16 of 26 Ph+ ALL patients showed discordant results in 27% of cases (32 of 117). Real-time quantitative PCR analysis of 19 ddPCR-positive samples with a low tumor burden failed to provide quantitative results in 63% of cases (12 of 19). These results highlight that in p190+ ALL the ddPCR method has a sufficient analytical performance for very low MRD monitoring and for predicting molecular relapse several months before hematologic relapse. In conclusion, MRD monitoring by ddPCR may better stratify Ph+ ALL patients at risk of disease progression.

For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology. Finally, after defining the BCR-ABL1 genomic junction, we performed the target CML patient-specific quantification, using droplet digital PCR (ddPCR). FISH and MinION steps, respectively, together with ddPCR analysis, greatly reduce the complexity that has impeded the use of "personalized monitoring" of CML in clinical practice. Our report suggests a feasible pipeline, in terms of costs and reproducibility, aimed at characterizing and quantifying the genomic BCR-ABL1 rearrangement during MRD monitoring in CML patients.

We report a third-generation sequencing assay on nanopore technology (MinION) for detecting BCR-ABL1 KD mutations and compare the results to a Sanger sequencing(SS)-based test in 24 Philadelphia-positive (Ph+) leukemia cases. Our data indicates that MinION is markedly superior to SS in terms of sensitivity, costs and timesaving, and has the added advantage of determining the clonal configuration of multiple mutations. We demonstrate that MinION is suitable for employment in the hematology laboratory for detecting BCR-ABL1 KD mutation in Ph+ leukemias.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid disorders characterized by ineffective hematopoiesis and an increased risk of transformation to acute myeloid leukemia (A...

The molecular pathogenesis of hematological diseases is often driven by genetic and epigenetic alterations. Next-generation sequencing has considerably increased our genomic knowledge of these disorders becoming ever more widespread in clinical practice. In 2012 Oxford Nanopore Technologies (ONT) released the MinION, the first long-read nanopore-based sequencer, overcoming the main limits of short-reads sequences generation. In the last years, several nanopore sequencing approaches have been performed in various "-omic" sciences; this review focuses on the challenge to introduce ONT devices in the hematological field, showing advantages, disadvantages and future perspectives of this technology in the precision medicine era.

Myeloid neoplasms with eosinophilia and abnormalities of the PDGFRA gene can benefit from therapy with tyrosine kinase inhibitors, therefore revealing the PDGFRA rearrangement is essential to ensure the best choice of treatment. The most common PDGFRA partner is the FIP1L1 gene, generating the oncoprotein FIP1L1/PDGFRA (F/P). In the majority of cases the F/P fusion gene originates from intrachromosomal rearrangement at band 4q12, and occasionally from chromosomal translocations. In both cases, the interstitial chromosomal deletion of a region involving the CHIC2 gene has been reported, which is cryptic by conventional karyotyping but detectable by Fluorescence In Situ Hybridization (FISH) analyses. Herein, we report an acute myeloid leukemia (AML) case presenting with eosinophilia; the F/P fusion gene originated from a new, cryptic and complex intrachromosomal rearrangement of 4q12. Classical FISH assay revealed abnormal hybridization signals, but the presence of the F/P chimaeric gene was demonstrated by molecular analysis. We performed molecular characterization of the chromosomal rearrangement and targeted Next-Generation Sequencing (NGS) analysis with a myeloid gene panel, revealing the presence of pathogenic genomic variants affecting the TET2 and ETV6 genes. These mutations were present as subclones at the disease onset and their clone size increased at relapse.

Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis, all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.

Most acute promyelocytic leukemia (APL) patients express PML-RARA fusion; in rare cases, RARA is rearranged with partner genes other than PML. To date, only 2 patients presenting features similar to APL showing the RARG gene rearrangement have been described. We report an acute myeloid leukemia patient with morphology resembling APL without involvement of the RARA gene. Molecular and fluorescent in situ hybridization analyses excluded PML-RARA fusion and variant rearrangements involving RARA and RARG loci. Targeted next-generation sequencing showed EZH2- D185H mutation. As this mutation involved the region of interaction with DNA methyltransferases, we speculate an epigenetic alteration of genes involved in the APL-like phenotype. Expression analysis by droplet digital polymerase chain reaction revealed downregulation of the RARA and RARG genes. We hypothesize a novel mechanism of EZH2 function alteration, which may be responsible for an acute myeloid leukemia with APL-like phenotype featuring dysregulation of the RARA and RARG genes.

The BCR-ABL1 fusion on the Philadelphia (Ph) chromosome is a hallmark of chronic myeloid leukemia (CML). More than 95 % of BCR-ABL1 transcripts in CML are either e13a2 or e14a2 (major breakpoint cluster region or M-bcr), whereas rare BCR-ABL1 transcripts are occasionally observed, accounting for less than 1 % of CML cases. Among these, a very rare fusion transcript joining the first 6 exons of BCR to exon 2 of ABL1 (e6a2) has been reported in various hematological malignancies characterized by an aggressive clinical course. We report a new case of blast crisis (BC) CML with an e6a2 fusion transcript characterized by many eosinophil precursors with abnormal granules. Moreover, fluorescence in situ hybridization analysis revealed genomic deletions of 1.3 megabases and 342 kilobases on der(9) of chromosome 9 and 22 sequences, respectively. The fusion transcript was quantified at diagnosis and during follow-up using digital droplet polymerase chain reaction (ddPCR) technology. The patient was treated with Dasatinib (140 mg/day), resulting in a 3-log reduction of the e6a2 transcript molecular burden from the third month after treatment. In this twentieth e6a2 case, characterized by marked eosinophilic dysplasia, deletions on der(9), and responsive to tyrosine kinase inhibitors therapy, we demonstrate that for molecular response monitoring of rare fusion transcripts associated with CML, ddPCR is a very useful technology.

Nested RT-PCR (nPCR) and real-time quantitative PCR (qPCR) are well-established methods for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL). Despite their remarkable sensitivity and specificity, both methods have inherent limitations, such as qualitative MRD evaluation and relative quantification. Herein, we used droplet digital PCR (ddPCR) to monitor MRD in 21 APL patients and compared its performance with nPCR and qPCR. After assessing the limit of detection (LOD) for each technique on serial dilutions of PML-RARA bcr1 and bcr3 transcripts, a total of 48 follow-up samples were analyzed and the results compared. ddPCR showed good linearity and efficiency and reached an LOD comparable or even superior to nPCR and qPCR. When tested on primary samples, ddPCR exhibited a sensitivity and specificity of ≥95% and ≥91% for bcr1 and bcr3 transcripts and displayed a significant concordance with both techniques, particularly with nPCR. The peculiar advantage of ddPCR-based monitoring of MRD is represented by absolute quantification, which provides crucial information for the management of patients whose MRD fluctuates under the LOD of qPCR and is detectable, but not quantifiable, by nPCR. Our findings highlight ddPCR as a reliable complementary approach to monitor MRD in APL, and suggest its advantageous application, particularly for the molecular follow-up of patients at high risk of relapse.

Background The assessment of TP53 mutational status is becoming a routine clinical practice for chronic lymphocytic leukemia patients (CLL). A broad spectrum of molecular techniques has been employed so far, including both direct Sanger sequencing and next generation sequencing. Oxford Nanopore Technologies recently released the MinION an USB-interfaced sequencer. In this paper we report our experience, with the MinION technology for the detection of the TP53 gene mutation in CLL patients. Twelve CLL patients at diagnosis were included in this study. All except one patient showed the TP53 gene deletion in Fluorescence in situ hybridization experiments. Patients were investigated for TP53 mutation by Sanger and by MinION sequencing. Analysis by Sanger was performed according with the IARC protocol. Analysis by MinION was performed adopting a strategy based on long template PCR, read error correction, and post variant calling filtering. Results Due to the high error rate of nanopore technology, sequence data were both used directly and before correction with two different in silico methods: ALEC and nanocorrect. A mean error rate of 15 % was detected before correction that was reduced to 4-5 % after correction. Analysis by Sanger sequencing was able to detect four patients mutated for TP53. MinION analysis detected one more mutated patient previously not detected from Sanger. Conclusion In our hands, the Nanopore technology shows correlation with Sanger sequencing but more sensitive, manageable and less expensive, and therefore has proven to be a useful tool for TP53 gene mutation detection. Electronic supplementary material The online version of this article (doi:10.1186/s13000-016-0550-y) contains supplementary material, which is available to authorized users.

Primary plasma cell leukemia (pPCL) is an uncommon form of plasma cell dyscrasia, and the most aggressive of the human monoclonal gammopathies. The t(11;14)(q13;q32) rearrangement is the most common alteration in pPCL, promoting cyclin D1 (CCND1) gene overexpression caused by its juxtaposition with the immunoglobulin heavy locus chromosome region. The myeloma overexpressed (MYEOV) gene maps very close to the CCND1 gene on chromosome 11, but its overexpression is rarely observed in multiple myeloma. The present study describes a case of pPCL with t(11;14) characterized by a breakpoint on der(11), unlike the one usually observed. Droplet digital polymerase chain reaction analysis revealed overexpression of CCND1 and MYEOV. To the best of our knowledge, MYEOV gene overexpression has never been previously described in pPCL.

Atypical chronic myeloid leukemia (aCML) is a rare hematological malignancy with a relative incidence estimated at 1–2 cases per 100 patients with BCR–ABL1-positive CML. The 2008 World Health Organ...

Acute myeloid leukemia (AML) is a hematologic disease with heterogeneous clinical and biological features. Although 40 – 50% of patients with AML carry an apparently normal karyotype (NK-AML), several molecular lesions have been found to be associated with this entity, thus improving stratifi cation of patients with NK-AML into prognostic risk groups [1]. Recent studies of cases of NK-AML by next-generation sequencing technologies have led to the identifi cation of new recurrent mutations in the BCL6 corepressor ( BCOR ) gene [2]. Th is gene is located on chromosome Xp11.4 and encodes a transcriptional corepressor with a nuclear localization. BCOR is a pivotal transcriptional regulator of early embryonic development, mesenchymal stem cell function and hemopoiesis [3]. Constitutional inactivating mutations of BCOR have been reported in the oculo-facial-cardiodental (OFCD) syndrome, which has an X-linked inheritance pattern [4]. In NK-AML, BCOR mutations occur in 4% of patients, being distributed along the whole coding sequence, associated with decreased transcript levels, absence of the full-length protein, and lacking/low expression of a truncated BCOR protein [2]. Moreover, the mutations target the functional allele in both male and female patients with AML. Th ese fi ndings support a role played by BCOR as a tumor-suppressor gene, inactivated by mutations in a subset of AML. Moreover, the detection of BCOR mutations seems to confer a poorer prognosis in terms of overall and event-free survival. Recently, BCOR mutations have been described in 4.2% of 354 and in 7.4% of 54 patients aff ected by myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML), respectively [5]. Patients with MDS with truncating mutations of BCOR had an inferior overall survival and a higher cumulative incidence of AML transformation [5]. We describe a case of AML with t(X;3) (p11.4;q22.1) showing down-regulated BCOR gene expression. Our fi ndings disclose a novel mechanism for BCOR gene inactivation, unlike those previously reported based on gene point mutations.

Introduction. Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. They are located in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu repeats methylation has been associated with tumor aggressiveness and investigated in some solid tumors, but the global Alu methylation level has not yet been investigated in hematological malignancies. Moreover, today, some of the techniques designed to measure global DNA methylation are focused on the methylation level of specific genomic compartments, including repeat elements. In this work we propose a new method for investigating Alu differential methylation, employing droplet digital PCR (ddPCR) technology, applied in patients affected by chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Methods. The study included a total of 46 patients: 30 CLL patients, 7 patients with MDS at intermediate/high risk, and 9 CMML patients. The study also involved acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with azacytidine (AZA) 0.75 µM or decytabine (DEC) 0.75 µM. Four healthy donors (HD) were also included as controls. For each DNA sample two aliquots of 250ng of gDNA were simultaneously digested (with 1 unit of Alu-in/sensitive isoschizomers either MspI or HpaII) and ligated (to a previously prepared synthetic adaptor) in parallel in two separate tubes. Considering that the genomic DNA amount in a human diploid cell is about 6 pg/cell, for each sample we calculated the percentage of methylated consensus Alu sequences as the ratio between the sum of positive droplets obtained from the three wells of both HpaII (MH) and MspI (MM) final dilutions, according to the following formula: [1-(sumMH/sumMM)]x100. The significance level was set at p Results. Using our ddPCR assay, we observed a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, and a minor decrease with AZA (p=0.058). Moreover, comparing the global Alu methylation levels at diagnosis and after AZA treatment in MDS patients, we observed a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis. We also extended the assessment of our assay in CLL patients at diagnosis. We observed a significant decrease of the Alu methylation level in CLL patients compared to HD. CLL patients were also classified in the following three cytogenetic risk groups according to the karyotypic alterations identified by Fluorescent In Situ Hybridization (FISH): low (with isolated 13q deletion), intermediate (without 11q, 13q and 17p deletions or with trisomy 12), and high risk (with 11q or, 17p deletions, or more than two chromosomal aberrations). Alu methylation status of the low and high-risk groups was more significantly reduced compared to HD, whereas considering intermediate-risk patients the difference was less evident. Finally, for CMML patients, a significant decrease of Alu sequences methylation was observed in patients harboring the main SRSF2 gene hotspot. However, these preliminary results should be confirmed by extending the analysis to other CMML patients. Conclusions. In our work, we propose a new method to investigate Alu differential methylation based on ddPCR technology. This assay represents an alternative to conventional quantitative-PCR (qPCR), introducing ddPCR as a more sensitive and immediate technique for Alu methylation analysis. Moreover, compared to qPCR, our ddPCR Alu assay may be carried out using very small amounts of digested gDNA (about 6 pg), and does not require a reference gene for the analysis of ddPCR data. To date, this is the first application of ddPCR to study global DNA methylation by inspecting DNA repeats. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose. Disclosures No relevant conflicts of interest to declare.

The problem of morbidity in deployed military personnel represents a much-debated topic. Because there have been two cases of thyroid cancer in the Tuscania regiment, the aim of the present study was to investigate the prevalence of all types of thyroid disease in a cohort of carabineers. A total of 673 carabineers, including 501 deployed carbineers (DCs) (29-48 years of age) and 172 nondeployed carabineers (NDCs) (29-51 years of age), of the Tuscania regiment were involved in the study. Thyroid volume, percentages of single nodules and multinodular goiter, percentage of autoimmune thyroid disease, and percentages and histological types of thyroid cancer were all measured. No statistical difference between DCs and NDCs was found for any of the data. Furthermore, when we divided DCs into subgroups according to time spent on deployment and time elapsed since the first deployment, we found no differences. However, a high prevalence of thyroid cancer was found in our cohort (2.0% in DCs and 2.5% in NDCs; not significant), and the prevalence of thyroid cancer in nodules in the cohort of carabineers was higher (10.0%) than the prevalence of thyroid cancer in nodules in the civilian population (5.6%, p < 0.001). No differences regarding the prevalence of thyroid diseases were observed when we compared DCs and NDCs, which suggests that no significant difference in exposure to toxic or carcinogenic substances that could have affected the thyroid occurred during deployments. The high prevalence of thyroid cancer in carabineers may merely reflect an increase of this cancer in the general population, or it may suggest the presence of some carcinogenic event in this specific cohort.

To report data regarding treatment with use of percutaneous laser ablation (PLA) in autonomously functioning thyroid nodules (AFTN).We treated 18 patients (10 women and 8 men, 31 to 80 years old) who presented with a single hyperfunctioning thyroid nodule (8 patients) or a multi-nodular goiter (10 patients) with clearly hyperfunctioning areas on a thyroid scintiscan. In 5 cases, free thyroxine (FT4) and free triiodothyronine (FT3) levels were high, and in these patients and a further 9 patients with cardiovascular symptoms, methimazole therapy was initiated to restore euthyroidism. The total number of PLA sessions ranged from 1 to 5 (median, 3). Thyroid-stimulating hormone, FT4, FT3, thyroglobulin, and antithyroglobulin and anti-thyroid peroxidase antibodies were measured by a commercial kit the day after PLA treatment, then weekly during the first month, and monthly thereafter. The Student t test was used for statistical analyses, and data are reported as mean values +/- SE.After each PLA session, there was a transient and mild increase in FT4 and FT3-5.2% to 18.1% (mean, 11.1 +/- 0.69%) (P0.001) in patients not treated with methimazole and 4.0% to 8.3% (mean, 5.9 +/- 0.31%) (P0.001) in patients treated with methimazole-relative to values before treatment; however, these values never reached the range of hyperthyroidism. In addition, thyroglobulin showed a remarkable increase after 24 hours- 115% to 390% (mean, 266.0 +/- 12.7%) (P0.001). Thyroid-stimulating hormone increased in all cases and reached normal values in all patients with single AFTN and in 5 patients (50%) with multinodular goiter within 3 months after PLA. At 1-year follow-up, the decrease in nodular volume was 24% to 72% (mean, 59.3 +/- 8.2%; P0.001).Our data show that PLA can be a useful treatment in AFTN and particularly in single toxic nodules. Possible elective indications are patients who refuse surgical or radioiodine treatment and patients with cardiovascular comorbidity who need rapid restoration of the euthyroid state and who cannot tolerate the discontinuation of antithyroid drugs for radioiodine treatment.

// Francesco Albano 1 , Antonella Zagaria 1 , Luisa Anelli 1 , Nicoletta Coccaro 1 , Giuseppina Tota 1 , Claudia Brunetti 1 , Crescenzio Francesco Minervini 1 , Luciana Impera 1 , Angela Minervini 1 , Angelo Cellamare 1 , Paola Orsini 1 , Cosimo Cumbo 1 , Paola Casieri 1 , Giorgina Specchia 1 1 Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology Section, University of Bari, 70124, Bari, Italy Correspondence to: Francesco Albano, e-mail: francesco.albano@uniba.it Keywords: acute promyelocytic leukemia, PML-RARA, relapse risk, droplet digital PCR, prognostic factor Received: February 25, 2015 Accepted: April 06, 2015 Published: April 18, 2015 ABSTRACT In this study we performed absolute quantification of the PML-RARA transcript by droplet digital polymerase chain reaction (ddPCR) in 76 newly diagnosed acute promyelocytic leukemia (APL) cases to verify the prognostic impact of the PML-RARA initial molecular burden. ddPCR analysis revealed that the amount of PML-RARA transcript at diagnosis in the group of patients who relapsed was higher than in that with continuous complete remission (CCR) (272 vs 89.2 PML-RARA copies/ng, p = 0.0004, respectively). Receiver operating characteristic analysis detected the optimal PML-RARA concentration threshold as 209.6 PML-RARA /ng (AUC 0.78; p 209.6 PML-RARA /ng had a worse relapse-free survival ( p = 0.0006). At 5-year follow-up, patients with >209.6 PML-RARA /ng had a cumulative incidence of relapse of 50.3% whereas 7.5% of the patients with suffered a relapse ( p < 0.0001). Multivariate analysis identified the amount of PML-RARA before induction treatment as the sole independent prognostic factor for APL relapse. Our results show that the pretreatment PML-RARA molecular burden could therefore be used to improve risk stratification in order to develop more individualized treatment regimens for high-risk APL cases.

OBJECTIVE To evaluate whether analysis of thyroid hormones in fine-needle aspiration (FNA) of thyroid nodules can provide information about the functional status and the nature of the nodules. METHODS We studied 4 groups of patients: group 1, 17 patients with autonomous hyperfunctioning thyroid nodules; group 2, 52 patients with cold nonfunctioning thyroid nodules; group 3, 12 patients with malignant thyroid nodules; and group 4 (control group), 10 patients with nonthyroid nodular lesions (enlarged parathyroid glands or lymph nodes). The assay of thyroid hormones was performed in FNA after the washing of needles and, with patient consent, also in normal thyroid parenchyma. RESULTS The free thyroxine (FT(4)) and free triiodothyronine (FT(3)) values were remarkably high in group 1 (mean, 5.5 +/- 0.53 ng/dL and 27.6 +/- 3.1 pg/mL, respectively; P

Electronic supplementary material The online version of this article (doi: 10.1007/s00277-016-2739-2 ) contains supplementary material, which is available to authorized users.

Patients affected by monoclonal gammopathy of undetermined significance (MGUS) very rarely develop a myelodysplastic syndrome (MDS). However, it was also demonstrated that MGUS patients had a significantly increased risk of developing MDS compared to the general population. We report a case of 5q-syndrome following a MGUS IgMk with mutation of MYD88 L256P. To our knowledge, this is the first case of del(5q) MDS following MGUS IgMk with the MYD88 L256P mutation in which there is coexistence of the markers of the two clonal diseases, but as an expression of distinct pathological features.

Introduction. BCR-ABL1 tyrosine kinase inhibitors (TKIs) are considered an important component of treatment for adult patients affected by Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). In fact, recent studies reported that treating Ph+ ALL with the combination of imatinib and multi-agent chemotherapy improved the overall outcome. Currently, no data are available on the impact of TKIs on minimal residual disease (MRD) in Ph+ ALL. In fact, although the real-time quantitative PCR (RQ-PCR) method, usually employed for monitoring the BCR-ABL1 residual transcript, is sensitive and easy to perform, it lacks a full standardization and international quality validation. Here, we describe a highly sensitive and reproducible droplet digital PCR (ddPCR) test to monitor BCR-ABL1 transcript level in Ph+ ALL. Methods. BCR-ABL1 expression analysis by ddPCR was performed in twenty-two newly diagnosed adult Ph+ ALL patients.The diagnosis was confirmed by qualitative RT-PCR specific for the BCR-ABL1 p190 fusion gene detection. ddPCR experiments were successfully performed in all twenty-two patients at the onset; several follow-up points were evaluated in thirteen patients. ddPCR experiments were performed using primers and probes specific for BCR-ABL1 p190. GUSB was used as control gene. Fifty ng and 750 ng of cDNA templates were used for the onset and for the post-treatment samples, respectively. To increase the limit of detection (LOD), three replicates were run for the post-treatment samples. ddPCR experiments were performed by Bio-Rad's QX200 system and ddPCR data were analyzed with QuantaSoft analysis software (version 1.7.4). Target concentration was expressed as BCR-ABL1 copies/mg. Results. First, we defined the LOD of the BCR-ABL1 p190 ddPCR system, a 10-fold dilution series (100, 10-1, 10-2, 10-3, 10-4, and 10-5) of a pool of p190 positive patients using a diluent-pool of healthy volunteers. This analysis showed remarkable linearity, trueness, and precision down to 10-5. After converting to log-log scale, linear regression showed no concentration-dependent bias, and R2 equaled 0.996. Because the negative samples showed no background, even the detection of a single droplet per well was considered a positive result. The median concentration of the BCR-ABL1 transcript at the onset was 233.8 (min 3.24 - max 1744) x 103BCR-ABL1 copies/mg. Concerning the analysis of follow-up samples, among the thirty-four points that were negative to qualitative nested RT-PCR, twenty-three (68%) resulted to be positive by ddPCR analysis, with a median concentration of 44.95 (min 0.27 - max 573.3) BCR-ABL1 copies/mg. Follow-up points that were negative in ddPCR remained negative even when the experiments were repeated increasing the depth of the analysis, evaluating a total quantity of 4.5 mg of RNA. Conclusions. This study indicates that, as compared to RQ-PCR, ddPCR increases the depth of the quantitative analysis of BCR-ABL1 p190 fusion transcript by allowing the evaluation of larger amounts of RNA. Moreover, our preliminary data revealed that the amount of the BCR-ABL1 fusion transcript at diagnosis is heterogeneous and that the ddPCR is much more sensitive than nested qualitative RT-PCR analysis, as the 68% of samples negative to nested PCR during the follow-up resulted to be positive by ddPCR. Therefore, we suggest that ddPCR represents a precise, sensitive and rapid method for both diagnosis and MRD monitoring of Ph+ ALL patients. Disclosures No relevant conflicts of interest to declare.

Recombinant human TSH (rhTSH) can be used for post-surgical radioiodine (I-131) thyroid remnants ablation in differentiated thyroid cancer (DTC) patients after surgery. Debate exists in literature about the optimal amount of I-131 that should be given for obtaining an effective ablation and about the role of iodine pool during treatment. Therefore, the aim of the present study was to assess whether I-131 ablation during rhTSH stimulus can be improved by reducing the circulating iodine pool and by increasing thyroid cell uptake and retention of I-131 obtained by administering furosemide and lithium.A total of 201 consecutive DTC patients were entered in the study: they were treated by total thyroidectomy and I-131 therapy during rhTSH stimulus to ablate thyroid remnants. Patients were divided into two groups according to the TNM stage: group 1 included patients in stage I-II who were treated with a low 30-mCi I-131 dose, while group 2 included patients in stage III-IV who were treated by a high 100-mCi I-131 dose. Moreover, both groups were further subdivided into three subgroups. Subgroup (a) included 45 patients from group 1 and 22 from group 2: they were treated with I-131 under rhTSH stimulus, following a short 4-day withdrawal of L-thyroxine (LT4). Subgroup (b) included 45 patients from group 1 and 22 from group 2: they were treated with I-131 under rhTSH stimulus, following a short 4-day withdrawal of L-T4, and after furosemide administration (25 mg/day orally) during the 3 days before I-131. Subgroup (c) included 45 patients from group 1 and 22 from group 2: they were treated with I-131 under rhTSH stimulus, following a short 4-day L-T4 withdrawal, and after administration of furosemide (25 mg/day orally) during the 3 days prior I-131 and lithium (450 mg/day orally) during the 3 days following I-131. Another group (group 3) of 20 patients characterized by a very low-risk cancer (unifocal tumor1.0 cm in diameter, without extra-capsular extension, N0) was treated with a 30-mCi I-131 dose under rhTSH stimulus without performing the short 4-day L-4 withdrawal: this group was taken as the control. Follow-up was performed by neck ultrasonography (US), and Tg measurement and I-131 WBS under rhTSH stimulus.Among the patients from group 1, those pre-treated with furosemide or with furosemide plus lithium showed a better outcome of ablation both in terms of undetectable Tg values (97.7% and 95.5 % vs. 79.5%, p0.05) and of WBS negativity (97.7% vs. 81.8%, p0.05) during the rhTSH stimulus. No similar findings were observed in group 2 patients. Moreover, in patients from group 3 (I-131 30 mCi, without L-T4 withdrawal), the outcome of ablation was significantly lower in comparison to patients from group 1 (I-131 30 mCi, with L-T4 withdrawal) in terms of undetectable Tg during the rhTSH stimulus (55.0%, p0.001).rhTSH is highly effective for post-surgical thyroid remnant ablation in low-risk cancer patients using the low 30-mCi dose protocol combined with the short 4-day withdrawal of L-T4. Moreover, in these patients the pre-treatment with furosemide seems to play an important role to further improve the outcome of ablation by reducing the iodine pool.

Objective To investigate whether recombinant human thyroid-stimulating factor (rhTSH) is effective for the radiometabolic ablation of post-surgery thyroid remnants, using low doses of 131 I. Patients and methods The study included two groups of patients enrolled consecutively: group 1 consisted of 52 patients with papillary cancer or minimally invasive follicular cancer (stage I and II), and group 2 consisted of 41 patients with the same stage of disease. All patients underwent a total thyroidectomy. Group 1 received 1.11 GBq (30mCi) 131 I for post-surgical remnants ablation with the aid of rhTSH, while group 2, in the hypothyroid state, received the same amount of radioiodine. To minimize iodine interference, all patients remained on a low iodine diet for 2 weeks and L-thyroxine (L-T4) was stopped for 4 days in the group of patients treated with the aid of rhTSH. To investigate 131 I uptake in this group, a tracer dose was administered 3 h after the second injection of rhTSH and the uptake was evaluated at 24 h just before administration of the therapeutic dose. 131 I was also measured in the patients treated in the hypothyroid state just before the therapeutic dose was given. Results After 1 year both groups were studied by using whole-body scintigraphy (WBS) and measuring thyroglobulin after rhTSH. In group 1, WBS was negative in 76.9% (40 patients), while thyroglobulin-stimulated levels were

The main steps in the management of differentiated thyroid cancer are thyroidectomy, treatment with iodine-131 ((131)I), and follow-up with whole-body scanning (WBS) and serum thyroglobulin (Tg) determination. Both (131)I treatment and follow-up require maximum stimulation of normal or pathological thyroid remnants by TSH. The use of recombinant human TSH (rhTSH) has been shown to be useful for follow-up, whereas previous reports are not univocal regarding the use of (131)I postsurgical ablation of thyroid remnants, at least when low doses (30 mCi) of (131)I are administered. A possible explanation for the diminished effectiveness of (131)I treatment after rhTSH may be the interference of iodine content of L-thyroxine (L-T4) therapy during the protocol of administration of rhTSH. We have evaluated the effectiveness of stimulation by rhTSH for radioiodine ablation of postsurgical remnants, stopping L-T4 the day before the first injection of rhTSH and restarting L-T4 the day after (131)I. The study included two groups of patients: group 1 included 16 patients with differentiated thyroid cancer (15 papillary cancers and 1 follicular cancer, stages I and II), who were treated with 30 mCi (131)I with the aid of rhTSH, using the standard protocol but stopping L-T4 as stated previously; and group 2 included 24 patients with the same features (histology and stage) of disease treated with 30 mCi in the hypothyroid state after L-T4 withdrawal. In both groups, serum TSH reached a very good stimulation level [76-210 U/liter (mean, 112 +/- 11 SE) and 38-82 U/liter (mean, 51 +/- 3 SE), respectively]. At the first WBS (after (131)I treatment), all patients showed thyroid remnants. Furthermore, two patients of the first group and three patients of the second group showed lymph node metastases. After 1 yr, all patients were studied again and underwent WBS with a tracer dose of (131)I and serum Tg measurement using rhTSH with the same protocol in both groups. The percentage of ablation (undetectable Tg and a negative WBS) was higher, although not reaching statistical significance, in patients treated with rhTSH: 81.2% in patients treated by rhTSH withdrawal and 75.0% in patients treated by L-T4 withdrawal, respectively. No patient experienced symptoms of hypothyroidism during the 4 d of L-T4 interruption, and serum T4 remained in the normal range. Urinary iodine was analyzed in both groups and compared with a control group of patients who received, for diagnostic purposes, rhTSH without stopping L-T4. In the first group, urinary iodine was 47.2 +/- 4.0 microg/liter (mean +/- SE; P = 0.21 vs. the second group, P = 0.019 vs. control group). In the second group, urinary iodine was 38.6 +/- 4.0 microg/liter (mean +/- SE; P < 0.001 vs. control group); urinary iodine in the control group was 76.4 +/- 9.3 microg/liter (mean +/- SE). Our data show that rhTSH, as administered in the protocol stated previously, allows at least the same rate of ablation of thyroid remnants when low doses (30 mCi) of (131)I are used. The possible role of interference of iodine content in L-T4 is not surprising if we consider that the amount of iodine in 30 mCi is negligible (5 microg) compared with the amount of iodine content in a daily dose of T(4) ( approximately 50 microg). The cost of rhTSH seems modest compared with the high cost of complex therapeutic regimens in other areas of oncology and in consideration of the well-being of patients and of the high level of effectiveness of the treatment.

Objective To report a case of an obese patient who had undergone renal transplantation and who had subtherapeutic levels of serum cyclosporine after treatment with orlistat. Methods The clinical and laboratory findings are presented, and the few cases reported in the literature are reviewed. Results A 29-year-old woman had subtherapeutic plasma levels of cyclosporine after orlistat treatment (360 mg/day) was initiated. The subtherapeutic levels persisted even though orlistat was administered the recommended 2 hours before ingestion of cyclosporine and even though the dosage of orlistat was decreased to only 240 mg/day. Because an increase of body weight is common after organ transplantation, treatment with orlistat has been used. In such patients, however, six cases of reduced therapeutic plasma levels of cyclosporine have been reported. Although a drug-drug interaction has been suggested, this case suggests that the decreased plasma cyclosporine levels are due to reduced absorption of fats rather than a drug-drug interaction. Because this patient was unable to adhere to a low-fat diet, she experienced severe diarrhea, a factor that may have dramatically diminished the absorption of cyclosporine. Conclusion Adherence to a low-fat diet should be strongly recommended if orlistat is prescribed to patients taking cyclosporine. Moreover, strict surveillance of the plasma concentration of cyclosporine is important. (Endocr Pract. 2002;8:124-126)

To assess whether fine-needle aspiration (FNA) can be a diagnostic procedure for identification of malignant thyroid nodules when the smear reveals the cytologic (microfollicular) finding of "follicular neoplasm" (FN).We reviewed a group of 79 patients with FN who underwent surgical treatment and evaluated the possible significance of the cytologic features of anisokaryosis, amount of colloid, and nuclear overlapping (NO). We excluded from the study those patients with FN who had other highly suspicious cytologic features, such as sporadic grooves and sporadic pseudonucleoli.In our series of 79 patients with FN at FNA, we found anisokaryosis in 49.4%, scant or no colloid in 31.6%, and NO in 5.1%. Thyroid cancer (TC)--follicular cancer or follicular variant of papillary cancer--was found in six patients (7.6%): four patients (10.2%) with anisokaryosis and two patients (5.0%) without anisokaryosis (P0.5; chi2 test). All the patients with malignant lesions had scant or no colloid in the smears, and four of them were those with NO. Therefore, TC was found in 24.0% of nodules with scant or no colloid versus 0.0% of nodules with abundant colloid (P0.001; chi2 test), and TC was found in 100% of nodules with NO versus 2.6% of nodules without NO (P0.001; chi2 test).Because of the high prevalence of thyroid nodules and the frequency of FN at FNA, the number of unnecessary interventions may be very high. FNA cannot be diagnostic in FN because invasion of the tumor capsule and blood vessels can be evidenced only at histologic examination. Although it is common opinion that microfollicular nodules should be surgically treated, simple cytologic features can help select patients at low risk who can be monitored and reassessed clinically and by FNA. Patients with high-risk cytologic features such as NO should be advised that a total thyroidectomy may be necessary.

Introduction Lymphoid enhancer-binding factor 1 (LEF1) is a downstream effector of the Wnt/ β-catenin signaling pathway, which controls cell growth and differentiation. In normal hematopoiesis, LEF1 plays a pivotal role in the development of B- and T-lymphocytes as well as granulocyte progenitor cells. High LEF1 expression has been reported as a favorable prognostic marker in cytogenetically normal acute myeloid leukemia whereas it is associated with poor prognosis in adult B precursor acute lymphoblastic leukemia and in chronic lymphocytic leukemia. Moreover, marked downregulation of LEF1 is associated with disease progression in myelodysplastic syndromes. Given the functional role of LEF1 in hematopoiesis and its putative prognostic impact in several hematological malignancies, we evaluated the prognostic significance of LEF1 expression in adult acute promyelocytic leukemia (APL). Methods One hundred and three consecutive patients with newly diagnosed APL were observed and treated with ATRA-based regimens (AIDA04937 and AIDA2000 GIMEMA protocols), between January 1996 and December 2012. LEF1 expression was measured by real-time qPCR in 78 patients (median age 45 years, range 16 to 88 years) with sufficient available material from the primary cohort. Advanced relative quantification analysis was performed using LightCycler 480 Software 1.5.1, based on the ΔΔCt method. APL samples were dichotomized at the median value and divided into two expression groups: low LEF1 (39 patients) with LEF1 values below the median value (LEF1low) and high LEF1 (39 patients) with LEF1 values above the median value (LEF1high). Clinical and biological features of the two groups were compared. A p value Results Patients with LEF1high expression had lower white blood cell counts at baseline (1.8 vs 12.0 x109/L; p < 0 .0001), and were less likely to carry a FLT3-ITD than LEF1low patients (12.8% vs 35.9%, respectively, p=0.02). The association between LEF1low and the presence of FLT3-ITD was also confirmed when the 11 patients with FLT3-TKD were included among patients with FLT3 mutations (p=0.03), or the group of FLT3 wild type patients, as compared to those bearing FLT3-ITD (p=0.03). Early death (defined as death during induction treatment) occurred in nine cases (23%) of the LEF1low group versus no case in the LEF1high group (OR = 0.04; p= 0.002). LEF1low expression was associated with a high relapse risk score (53.9% vs 7.7%, OR=0.07; p < 0.0001). The LEF1high group showed a trend toward a statistically significant association with a lower median age (p = 0.08). No significant differences were observed regarding CD34, CD2, bcr3 positivity and LEF1 expression. Survival analysis of 61 APL patients < 60 years revealed that the LEF1high group had significantly longer overall survival (OS) (p = 0.03), whereas no differences were observed between the two groups in terms of relapse-free survival. Cox analysis for OS confirmed only LEF1 expression as an independent prognostic factor (HR=5.4; 95% CI, 1.0 – 27.5, p =0.04). Among the 17 patients over the age of 60, those with LEF1high expression showed a higher median survival than those in the LEF1low group (6.5 vs 0.04 y.rs, p = 0.05). In silico analysis of the differential expression of LEF1 in APL identified 9 differentially expressed, up-modulated genes associated with a high expression of LEF1 (ETS1, FAIM3, CCR7, IL7R, LCK, IL2RB, ITK, RASGRP1, TRBC1); the majority of these genes is involved in the regulation of apoptosis Conclusions Our study provides, for the first time, evidence that LEF1 expression is an independent prognostic factor in APL and that it could be used in patients risk stratification. The observation provided by in silico gene expression analysis that LEF1 expression in APL is associated with biologic changes, mostly in terms of apoptosis regulation, will need to be confirmed experimentally. Disclosures: No relevant conflicts of interest to declare.

Roberto Anichini,1 Sabrina Cosimi,2 Alberto Di Carlo,3 Paola Orsini,4 Alessandra De Bellis,1 Giuseppe Seghieri,1 Flavia Franconi,5 Fabio Baccetti6 1Diabetes Unit, Spedali Riuniti, Pistoia, Italy; 2Diabetes Unit, Hospital of Versilia, Camaiore (LU), Italy; 3Diabetes Unit, Hospital of Lucca, Italy; 4Diabetes Unit, Hospital of Livorno, Italy; 5Department of Biochemical Sciences, University of Sassari, Italy; 6Diabetes Unit, Hospital of Massa, Italy Purpose: To investigate whether gender affects therapeutic response by exenatide twice a day (BID) in type 2 diabetes by using a database concerning patients monitored by five outpatient clinics in Tuscany, Italy. Patients and methods: We considered a cohort of 315 (154 male/161 female) patients experiencing therapeutic failure while on oral therapy (metformin, or combination therapy metformin + sulphonylureas), who were given exenatide (10 µg/BID) and who fully completed 4 months, 8 months, and 12 months of follow-ups. Results: Among patients stratified by gender and well matched for age, body mass index, and hemoglobin A1c (HbA1c), it was found that the length of disease was longer in females than in males (12 ± 8 years versus 10 ± 7 years; P = 0.037), and the ratio of patients on metformin to those on combination therapy was higher in men (P = 0.018). Target glycemic response (1-year HbA1c ≤ 7%) was achieved in a significantly higher proportion of males than females (38% versus 27%;Χ2 = 4.66; P = 0.03). Target weight loss expressed as 1-year weight percent fall from baseline ≥ 75th percentile (8.5%) was significantly higher in females at 8 and 12 months (P < 0.05; for both). One-year glycemic target response was inversely related to baseline HbA1c levels and diabetes duration among males, while metformin therapy (compared to oral combination therapy) was a significant predictor of better glycemic targets among females. Homeostasis model assessment-B, measured in 117 patients, predicted hypoglycemic response only in women (P = 0.009). Target 1-year weight loss was predicted by longer diabetes duration among males and by lower baseline HbA1c among females. Finally, no significant difference between genders was noted as to gastrointestinal side effects after exenatide therapy. Conclusion: According to this “real world” experience, predictors of glycemic control and body weight loss after 12 months of exenatide BID therapy are different between genders in type 2 diabetes. Keywords: GLP-1 agonist therapy, exenatide BID, type 2 diabetes, real world setting

// Francesco Albano 1 , Antonella Zagaria 1 , Luisa Anelli 1 , Paola Orsini 1 , Crescenzio Francesco Minervini 1 , Luciana Impera 1 , Paola Casieri 1 , Nicoletta Coccaro 1 , Giuseppina Tota 1 , Claudia Brunetti 1 , Angela Minervini 1 , Domenico Pastore 1 , Paola Carluccio 1 , Anna Mestice 1 , Angelo Cellamare 1 , and Giorgina Specchia 1 1 Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology Section, University of Bari, Bari, Italy. Correspondence: Francesco Albano, email: // Keywords : acute promyelocytic leukemia, LEF1; prognosis; outcome. Received : November 20, 2013 Accepted : December 19, 2013 Published : December 19, 2013 Abstract Lymphoid enhancer-binding factor 1 ( LEF1 ) is a downstream effector of the Wnt/ β-catenin signaling pathway. High LEF1 expression has been reported as a prognostic marker in hematologic malignancies. We evaluated the prognostic significance of LEF1 expression in 78 adult acute promyelocytic leukemia (APL) patients. APL samples were dichotomized at the median value and divided into: LEF1 low and LEF1 high . LEF1 high patients had lower WBC counts at baseline and were less likely to carry a FLT3 -ITD than LEF1 low patients. Early death occurred only in the LEF1 low group. Moreover, LEF1 low expression was associated with a high Sanz score. Survival analysis of 61 APL patients < 60 years revealed that the LEF1 high group had a significantly longer overall survival (OS). Cox analysis for OS confirmed only LEF1 expression as an independent prognostic factor. Of the 17 patients over the age of 60, those in the LEF1 high group showed a higher median survival. In silico analysis identified 9 differentially expressed, up-modulated genes associated with a high expression of LEF1 ; the majority of these genes is involved in the regulation of apoptosis. Our study provides evidence that LEF1 expression is an independent prognostic factor in APL, and could be used in patients risk stratification.