A novel mutation of Twinkle in Perrault syndrome: A not rare diagnosis?

Perrault syndrome is a rare disorder characterized by ovarian dysgenesis, bilateral sensorineural hearing loss and associated with mutations in six mitochondrial proteins. Additional neurological features were also described. Herein, we report on a 27-year-old woman with Perrault syndrome (PS), moderate ataxia and axonal sensory-motor peripheral neuropathy in whom we identified compound heterozygous mutations in the TWNK gene (p.Val507Ile and the novel p.Phe248Ser variant). Fewer than 30 patients with PS have been reported worldwide. Neurological involvement is more frequently associated with mutations in TWNK and indicates possible genotype-phenotype correlations. TWNK mutations should be searched in patients with sensory ataxia, early onset bilateral sensorineural hearing loss, and ovarian dysfunction in women.

High feasibility and antileukemic efficacy of fludarabine, cytarabine, and idarubicin (FLAI) induction followed by risk-oriented consolidation: A critical review of a 10-year, single-center experience in younger, non M3 AML patients.

About 105 consecutive acute myeloid leukemia (AML) patients treated with the same induction-consolidation program between 2004 and 2013 were retrospectively analyzed. Median age was 47 years. The first induction course included fludarabine (Flu) and high-dose cytarabine (Ara-C) plus idarubicin (Ida), with or without gemtuzumab-ozogamicin (GO) 3 mg/m(2) (FLAI-5). Patients achieving complete remission (CR) received a second course without fludarabine but with higher dose of idarubicin. Patients not achieving CR received an intensified second course. Patients not scheduled for early allogeneic bone marrow transplantation (HSCT) where planned to receive at least two courses of consolidation therapy with Ara-C. Our double induction strategy significantly differs from described fludarabine-containing regimens, as patients achieving CR receive a second course without fludarabine, to avoid excess toxicity, and Ara-C consolidation is administrated at the reduced cumulative dose of 8 g/m(2) per cycle. Toxicity is a major concern in fludarabine containing induction, including the recent Medical Research Council AML15 fludarabine, cytarabine, idaraubicin and G-CSF (FLAG-Ida) arm, and, despite higher anti-leukemic efficacy, only a minority of patients is able to complete the full planned program. In this article, we show that our therapeutic program is generally well tolerated, as most patients were able to receive subsequent therapy at full dose and in a timely manner, with a 30-day mortality of 4.8%. The omission of fludarabine in the second course did not reduce efficacy, as a CR rate of 83% was achieved and 3-year disease-free survival and overall survival (OS) were 49.6% and 50.9%, respectively. Our experience shows that FLAI-5/Ara-C + Ida double induction followed by risk-oriented consolidation therapy can result in good overall outcome with acceptable toxicity. Am. J. Hematol. 91:755-762, 2016. © 2016 Wiley Periodicals, Inc.

Chronic myeloid leukemia: a prospective comparison of interphase fluorescence in situ hybridization and chromosome banding analysis for the definition of complete cytogenetic response: a study of the GIMEMA CML WP.

In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Establishment and Characterization of a Novel Fibroblastic Cell Line (SCI13D) Derived from the Broncho-Alveolar Lavage of a Patient with Fibrotic Hypersensitivity Pneumonitis.

Hypersensitivity pneumonitis (HP) is a diffuse interstitial lung disease (ILD) caused by the inhalation of a variety of antigens in susceptible individuals. Patients with fibrotic HP (fHP) may show histopathological and radiological manifestations similar to patients with idiopathic pulmonary fibrosis (usual interstitial pneumonia-like pattern of fibrosis) that are associated with a worse prognosis. We describe here the establishment and characterization of a fibroblastic cell line derived from the broncho-alveolar lavage (BAL) of a patient with fHP, a 53 year old man who presented at our Pneumology Unit with cough and dyspnea. The fHP diagnosis was based on international criteria and multidisciplinary discussion. Primary fibroblasts were expanded in vitro until passage 36. These fibroblasts displayed morpho/phenotypical features of myofibroblasts, showing high positivity for α-smooth muscle actin, type I collagen, and fibronectin as determined by quantitative RT-PCR and cyto-fluorographic analysis. Cytogenetic analyses further evidenced trisomy of chromosome 10, which interestingly harbors the FGF2R gene. To our knowledge, this is the first fibroblastic cell line derived from an fHP patient and might, therefore, represent a suitable tool to model the disease in vitro. We preliminarily assessed here the activity of pirfenidone, further demonstrating a consistent inhibition of cells growth by this antifibrotic drug.

Doxorubicin induces senescence or apoptosis in rat neonatal cardiomyocytes by regulating the expression levels of the telomere binding factors 1 and 2.

Low or high doses of doxorubicin induce either senescence or apoptosis, respectively, in cardiomyocytes. The mechanism by which different doses of doxorubicin may induce different stress-response cellular programs is not well understood. A recent study showed that the level of telomere dysfunction may induce senescence or apoptosis. We investigated the pathways to both apoptosis and senescence in neonatal rat cardiomyocytes and in H9c2 cells exposed to a single pulsed incubation with low or high doses of doxorubicin. High-dose doxorubicin strongly reduces TRF2 expression while enhancing TRF1 expression, and it determines early apoptosis. Low-dose doxorubicin induces downregulation of both TRF2 and TRF1, and it also increases the senescence-associated-beta-galactosidase activity, downregulates the checkpoint kinase Chk2, induces chromosomal abnormalities, and alters the cell cycle. The involvement of TRF1 and TRF2 with apoptosis and senescence was assessed by short interfering RNA interference. The cells maintain telomere dysfunction and a senescent phenotype over time and undergo late death. The increase in the phase>4N and the presence of micronuclei and anaphase bridges indicate that cells die by mitotic catastrophe. p38 modulates TRF2 expression, whereas JNK and cytoplasmic p53 regulate TRF1. Pretreatment with specific inhibitors of MAPKs and p53 may either attenuate the damage induced by doxorubicin or shift the cellular response to stress from senescence to apoptosis. In conclusion, various doses of doxorubicin induce differential regulation of TRF1 and TRF2 through p53 and MAPK, which is responsible for inducing either early apoptosis or senescence and late death due to mitotic catastrophe.

HMGA2 overexpression in polycythemia vera with t(12;21)(q14;q22).

Chromosomal translocations involving the 12q14 band are rarely detected in hematological disorders, and are usually correlated with HMGA2 gene expression. HMGA2 is highly expressed during embryonic cell growth and differentiation, and regulates transcription and chromatin organization, but is rarely detectable in adult tissues. We describe a case of polycythemia vera with a t(12;21)(q14;q22). The 12q14 breakpoint was characterized by fluorescence in situ hybridization analysis using the bacterial artificial chromosome RP11-366L20 containing 3' sequences of the HMGA2 gene. Qualitative and quantitative polymerase chain reaction showed the presence of high levels of HMGA2 gene expression, which were temporarily reduced with hydroxyurea therapy. The present case confirms that involvement of the 12q14 band may be associated with HMGA2 overexpression in chronic Philadelphia chromosome-negative myeloproliferative disease, regardless of the partner chromosome involved in the translocation. Such overexpression may contribute to the pathogenesis of the disease, which otherwise of itself shows a favorable and stable course.

Molecular analysis of the Imatinib-induced complete cytogenetic response in chronic myelogenous leukemia.

The aim of this study was to elucidate the relationship between clonal and normal haematopoiesis in chronic myelogenous leukemia (CML). Thirteen female patients who reached complete cytogenetic response (CCR) after Imatinib treatment were studied. Although all the study patients exhibited a polyclonal pattern of X inactivation, p210 BCR/ABL transcript remained detectable in all cases by nested RT-PCR. This study also demonstrated the presence of p190 transcript in nine out of 13 study patients. A longer follow-up analysis is needed to clarify the prognostic value of these results. The recent observation that clonal cytogenetic abnormalities may occur in CML patients in polyclonal remission after imatinib suggests that a neoplastic stem cell lacking BCR/ABL rearrangement may acquire further cytogenetic alterations other than Philadelphia chromosome.

Masked Philadelphia chromosome due to atypical BCR/ABL localization on the 9q34 band and duplication of the der(9) in a case of chronic myelogenous leukemia.

The cytogenetic studies and molecular evaluation of a Philadelphia chromosome negative chronic myelogenous leukemia patient with trisomy 21 (100% metaphases) and trisomy 9 (50% metaphases) at diagnosis are described. Fluorescence in situ hybridization revealed an atypical location of the BCR/ABL fusion signal on 9q, which was duplicated in cells with trisomy 9 simulating a double Ph. The patient was successfully treated with Glivec (also known as Gleevec; Novartis, Basel, Switzerland) and achieved complete hematological and cytogenetic response as well as a reduction of BCR/ABL transcripts detected by real-time quantitative PCR.

Combined assessment of WT1 and BAALC gene expression at diagnosis may improve leukemia-free survival prediction in patients with myelodysplastic syndromes.

Several genes with relevant pathogenetic and prognostic value have been identified in patients with myelodysplastic syndromes. Overexpression of WT1 at diagnosis has been associated with increased progression to acute myeloid leukemia and reduced leukemia free survival. Conversely, few data are available on the prognostic value of BAALC gene overexpression in AML and MDS patients. We evaluated the prognostic value of the combined expression of WT1 and BAALC genes at diagnosis in 86 MDS patients who had been stratified according to IPSS and R-IPSS scoring systems. Our results suggest that in the whole group of patients, low levels of both WT1 and BAALC were associated with a favorable outcome (3-year LFS 74.5%, median not reached), whereas patients presenting high expression levels of both genes had the worst prognosis (3-year LFS 0%, median 12 months, p<0.001). More specifically, molecular profiling was especially useful for intermediate 1 and intermediate-2/high risk groups. This study suggests that evaluating WT1 and BAALC gene expression at diagnosis may improve standard risk stratification and possibly refine the therapeutic approach for MDS patients.

Standard and variant Philadelphia translocation in a CML patient with different sensitivity to imatinib therapy.

Most chronic myeloid leukemia (CML) patients show the Philadelphia chromosome (Ph) arising from the reciprocal t(9;22), but 5-10% present variants of this translocation involving different breakpoints besides 9q34 and 22q11. WE REPORT THE NON SIMULTANEOUS OCCURRENCE OF TWO DIFFERENT TYPES OF PH TRANSLOCATION IN A CML PATIENT: a t(9;22)(q34;q11) standard and a three-way variant t(9;11;22)(q34;p15;q11). Bone marrow cells with standard translocation did not have BCR/ABL kinase domain (KD) mutations and were sensitive to imatinib therapy. In contrast, bone marrow cells with the variant translocation showed two BCR/ABL KD mutations and were resistant to imatinib, thus inducing transformation to the blast phase and karyotype evolution.

Integrating post induction WT1 quantification and flow-cytometry results improves minimal residual disease stratification in acute myeloid leukemia.

Fifty uniformly treated adult AML patients were analyzed with respect to pre-treatment and post-induction risk factors. Forty-two patients achieving complete hematological remission were assessed for minimal residual disease (MRD) by WT1 gene expression; 34 by flow-cytometry (flow-MRD). Patients who were flow-MRD negative had a better 3-year disease-free (DFS; 79.5% vs. 27.3%; p=.032) compared with patients who were still positive after induction. Interestingly, DFS of flow-MRD positive patients was not related to the amount of flow-detected clone population (≥ or <1%, p=.41) but to WT1 reduction (ΔWT1, 3-year DFS; 46.2% vs. 0% if ΔWT1 was ≥ or < of 1.5 log, p=.001). In AML, combining MRD results provided by WT1 quantification and flow-cytometry improves the reliability of MRD-based prognostic stratification. Similar analyses by further larger studies should be advocated.

Evaluating treatment response of chronic myeloid leukemia: emerging science and technology.

Chronic myeloid leukemia (CML) is a hematological disease accounting for about 15-20% of all adult leukemias. The clinical and biologic advances achieved in such a malignancy, represent one of the best successes obtained by translational medicine. Indeed, identification of the fusion oncogene BCR-ABL has allowed using of small molecule inhibitors of its tyrosine kinase activity which, in turn, have literally revolutionized the treatment of CML. Importantly the successfully clinical management was also realized on appropriate diagnosis, disease monitoring as well as early identification of such mutations causing drug resistance. Notably the recent availability of refined laboratory equipments represented by the Next Generation Sequencing (NGS) and genomic analyses has further contributed to gain ground towards the cure of this tumor. These issues are discussed here together with an overview on how patients treated with tyrosine kinase inhibitors should be monitored.

p38 MAPK and JNK antagonistically control senescence and cytoplasmic p16INK4A expression in doxorubicin-treated endothelial progenitor cells.

Patients treated with low-dose anthracyclines often show late onset cardiotoxicity. Recent studies suggest that this form of cardiotoxicity is the result of a progenitor cell disease. In this study we demonstrate that Cord Blood Endothelial Progenitor Cells (EPCs) exposed to low, sub-apoptotic doses of doxorubicin show a senescence phenotype characterized by increased SA-b-gal activity, decreased TRF2 and chromosomal abnormalities, enlarged cell shape, and disarrangement of F-actin stress fibers accompanied by impaired migratory ability. P16( INK4A) localizes in the cytoplasm of doxorubicin-induced senescent EPCs and not in the nucleus as is the case in EPCs rendered senescent by different stimuli. This localization together with the presence of an arrest in G2, and not at the G1 phase boundary, which is what usually occurs in response to the cell cycle regulatory activity of p16(INK4A), suggests that doxorubicin-induced p16( INK4A) does not regulate the cell cycle, even though its increase is closely associated with senescence. The effects of doxorubicin are the result of the activation of MAPKs p38 and JNK which act antagonistically. JNK attenuates the senescence, p16( INK4A) expression and cytoskeleton remodeling that are induced by activated p38. We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity. In conclusion, this study provides a better understanding of the senescence of doxorubicin-treated EPCs, which may be helpful in preventing and treating late onset cardiotoxicity.

A BCR-JAK2 fusion gene as the result of a t(9;22)(p24;q11) in a patient with acute myeloid leukemia.

We report the occurrence of a BCR-JAK2 fusion gene in a case of acute myeloid leukemia (AML) resulting from a t(9;22)(p24;q11) translocation as the sole cytogenetic abnormality. The BCR-JAK2 fusion gene has the same breakpoint in BCR as is found in the BCR/ABL p210. The chimeric gene is the result of a reciprocal translocation between chromosomes 9 and 22, which implies a double break on chromosome 9; this has allowed generating an in-frame fusion transcript. Previously, BCR-JAK2 rearrangement was observed in a single case with atypical chronic myelogenous leukemia (CML), but in that case the breakpoint in the BCR was different.