Identification of genetic targets in acute myeloid leukaemia for designing targeted therapy.

Few effective therapies exist for acute myeloid leukaemia (AML), in part due to the molecular heterogeneity of this disease. We sought to identify genes crucial to deregulated AML signal transduction pathways which, if inhibited, could effectively eradicate leukaemia stem cells. Due to difficulties in screening primary cells, most previous studies have performed next-generation sequencing (NGS) library knockdown screens in cell lines. Using carefully considered methods including evaluation at multiple timepoints to ensure equitable gene knockdown, we employed a large NGS short hairpin RNA (shRNA) knockdown screen of nearly 5 000 genes in primary AML cells from six patients to identify genes that are crucial for leukaemic survival. Across various levels of stringency, genome-wide bioinformatic analysis identified a gene in the NOX family, NOX1, to have the most consistent knockdown effectiveness in primary cells (P = 5∙39 × 10-5 , Bonferroni-adjusted), impacting leukaemia cell survival as the top-ranked gene for two of the six AML patients and also showing high effectiveness in three of the other four patients. Further investigation of this pathway highlighted NOX2 as the member of the NOX family with clear knockdown efficacy. We conclude that genes in the NOX family are enticing candidates for therapeutic development in AML.

Repeat serological testing for anti-citrullinated peptide antibody after commencement of therapy is not helpful in patients with seronegative rheumatoid arthritis.

AIM: To Investigate the prevalence of seroconversion to ACPA after commencement of triple disease-modifying anti-rheumatic drug (DMARD) treat-to-target therapy. BACKGROUND: Anti-citrullinated peptide antibody (ACPA) and rheumatoid factor (RF) define 'seropositive' rheumatoid arthritis (RA). Both predict unfavourable disease course, development of extra-articular features and treatment outcomes. We investigated the prevalence of seroconversion to ACPA after commencement of triple disease-modifying anti-rheumatic drug (DMARD) treat-to-target therapy. METHODS: DMARD-naïve patients with RA according to the 1987 American College of Rheumatology criteria and disease duration of <96 weeks were enrolled. RF and ACPA levels were recorded at baseline and sequentially during triple DMARD therapy. RESULTS: A total of 368 patients were followed for a median of 272 weeks. Of 154 patients seronegative for ACPA at recruitment, 10 (6.5%) seroconverted at some point. Nine of these were positive for RF at baseline and baseline RF titre was predictive of seroconversion. Four (2.6%) patients remained seropositive. No patients seroconverted from negative to positive for both RF and ACPA. Median time to seroconversion for ACPA was 29 months. CONCLUSION: Persistent seroconversion of ACPA from negative to positive after diagnosis in patients with RA is uncommon. ACPA and RF double negative patients are highly unlikely to ever develop ACPA positivity with a risk <1%. It is therefore unlikely to be helpful or cost effective to perform serial ACPA measurements in patients with seronegative RA after commencement of a treat-to-target strategy.

A Comparison of Partial Discharge Sensors for Natural Gas Insulated High Voltage Equipment.

The research in this paper consists of practical experimentation on a gas insulated section of high voltage equipment filled with carbon dioxide and technical air as a direct replacement to sulphur hexafluoride (SF6) and analyses the results of PD measurement by way of internal UHF sensors and external HFCTs. The results contribute to ongoing efforts to replace the global warming gas SF6 with an alternative such as pure carbon dioxide or technical air and are applicable to mixtures of electronegative gases that have a high content of buffer gas including carbon dioxide. The experiments undertaken involved filling a full-scale gas insulated line demonstrator with different pressures of CO2 or technical air and applying voltages up to 242 kV in both clean conditions and particle contaminated conditions. The results show that carbon dioxide and technical air can insulate a gas section normally insulated with SF6 at phase-to-earth voltage of 242 kV and that both HFCT and UHF sensors can be used to detect partial discharge with natural gases. The internal UHF sensors show the most accurate PD location results but external HFCTs offer a good compromise and very similar location accuracy.

Mental Health and Disorders of Sex Development/Intersex Conditions in Iranian Culture: Congenital Adrenal Hyperplasia, 5-α Reductase Deficiency-Type 2, and Complete Androgen Insensitivity Syndrome.

Sixty-one patients (22 patients with congenital adrenal hyperplasia [CAH] with a mean age of 14.86 years [range, 5-23], 20 patients with 5-α reductase deficiency type 2 [5α-RD-2] with a mean age of 19.5 years [range, 5-29], and 19 patients with complete androgen insensitivity syndrome [CAIS] with a mean age of 18.26 years [range, 5-28]) were evaluated using the Kiddie Schedule for Affective Disorders and Schizophrenia, the Structured Clinical Interview for DSM-IV Axis I, Axis II, and the Global Assessment Functioning Scale. All participants were female-assigned at birth. Ten patients (16.4%) transitioned to the male gender. Overall, 68% of patients had one or more lifetime Axis I disorders, including 63.6% of the CAH participants, 90% of 5α-RD-2 participants, and 52.6% of the CAIS participants. The most commonly observed were affective disorders (27.9%), gender identity disorder (27.9%), and anxiety (16.4%). Our study demonstrates that mental health of Iranian patients with DSD is at risk. This might be due to the fact that patients with DSD conditions are mostly treated medically and their mental health is often superficially addressed in developing countries such as Iran, at least in the past. We argue that it is important to pay attention to the mental health issues of patients with DSD and focus on specific issues, which may vary cross-culturally.

Childhood Sex-Typed Behavior and Gender Change in Individuals with 46,XY and 46,XX Disorders of Sex Development: An Iranian Multicenter Study.

Disorders of sex development (DSD) are congenital conditions in which the typical genetic and hormonal profiles are affected and thereby the usual process of sexual differentiation. Most of these studies, however, have been conducted in Western countries. In the present study, preschool sex-typed activities of Iranian individuals with DSD and their age-matched non-affected male and female relatives were assessed using the Pre-School Activities Inventory (PSAI) modified for retrospective self-report. A total of 192 individuals participated in our study, including 33 46,XX individuals with congenital adrenal hyperplasia (CAH; M age = 10.36, SD = 5.52), 15 46,XY individuals with complete androgen insensitivity syndrome (CAIS; M age = 19.8, SD = 7.14), and 16 46,XY individuals with 5-alpha reductase deficiency type-2 (5α-RD-2; M age = 17.31, SD = 7.28), as well as one age-matched non-affected male and female relative for each patient. With regard to PSAI scores, male-identifying participants with 5α-RD-2 and male controls reported similar levels of male-typical childhood play. Female-identifying participants with 5α-RD-2 and CAH showed comparable scores: significantly less masculine and more feminine than male controls, but significantly more masculine and less feminine than females with CAIS and female controls. These findings support the role of androgens in the development of sex-typical childhood play behavior, with those being exposed to higher levels of fetal functional androgens expressing more masculine behavior at preschool ages.

RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1IS (%BCR-ABL1IS) by reverse transcription-quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification. METHODS: BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay. RESULTS: Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range. CONCLUSIONS: RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.

Combining BCR-ABL1 transcript levels at 3 and 6 months in chronic myeloid leukemia: implications for early intervention strategies.

Several groups have shown that that the BCR-ABL1 transcript level measured at 3 or 6 months after starting treatment with tyrosine kinase inhibitors strongly predicts clinical outcomes for patients with chronic myeloid leukemia. In this work, we asked whether the prognostic value of the 3-month transcript level could be improved by combining the 3- and 6-month results. We classified patients treated with imatinib and patients treated with dasatinib according to their transcript levels at 3 months and 6 months. The patients who met the 3-month landmark but failed the 6-month one had outcomes identical to those of patients who met both landmarks, whereas the patients who failed the first landmark but met the second one had prognoses similar to those who failed both landmarks. In summary, early intervention strategies can be based robustly just on the transcript level at 3 months. This trial was registered at www.clinicaltrials.gov as # NCT01460693.

Clinical drug resistance linked to interconvertible phenotypic and functional states of tumor-propagating cells in multiple myeloma.

The phenotype and function of cells enriched in tumor-propagating activity and their relationship to the phenotypic architecture in multiple myeloma (MM) are controversial. Here, in a cohort of 30 patients, we show that MM composes 4 hierarchically organized, clonally related subpopulations, which, although phenotypically distinct, share the same oncogenic chromosomal abnormalities as well as immunoglobulin heavy chain complementarity region 3 area sequence. Assessed in xenograft assays, myeloma-propagating activity is the exclusive property of a population characterized by its ability for bidirectional transition between the dominant CD19(-)CD138(+) plasma cell (PC) and a low frequency CD19(-)CD138(-) subpopulation (termed Pre-PC); in addition, Pre-PCs are more quiescent and unlike PCs, are primarily localized at extramedullary sites. As shown by gene expression profiling, compared with PCs, Pre-PCs are enriched in epigenetic regulators, suggesting that epigenetic plasticity underpins the phenotypic diversification of myeloma-propagating cells. Prospective assessment in paired, pretreatment, and posttreatment bone marrow samples shows that Pre-PCs are up to 300-fold more drug-resistant than PCs. Thus, clinical drug resistance in MM is linked to reversible, bidirectional phenotypic transition of myeloma-propagating cells. These novel biologic insights have important clinical implications in relation to assessment of minimal residual disease and development of alternative therapeutic strategies in MM.

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies.

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

Preclinical and clinical efficacy of XPO1/CRM1 inhibition by the karyopherin inhibitor KPT-330 in Ph+ leukemias.

As tyrosine kinase inhibitors (TKIs) fail to induce long-term response in blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL), novel therapies targeting leukemia-dysregulated pathways are necessary. Exportin-1 (XPO1), also known as chromosome maintenance protein 1, regulates cell growth and differentiation by controlling the nucleocytoplasmic trafficking of proteins and RNAs, some of which are aberrantly modulated in BCR-ABL1(+) leukemias. Using CD34(+) progenitors from CML, B-ALL, and healthy individuals, we found that XPO1 expression was markedly increased, mostly in a TKI-sensitive manner, in CML-BC and Ph(+) B-ALL. Notably, XPO1 was also elevated in Ph(-) B-ALL. Moreover, the clinically relevant XPO1 inhibitor KPT-330 strongly triggered apoptosis and impaired the clonogenic potential of leukemic, but not normal, CD34(+) progenitors, and increased survival of BCR-ABL1(+) mice, 50% of which remained alive and, mostly, became BCR-ABL1 negative. Moreover, KPT-330 compassionate use in a patient with TKI-resistant CML undergoing disease progression significantly reduced white blood cell count, blast cells, splenomegaly, lactate dehydrogenase levels, and bone pain. Mechanistically, KPT-330 altered the subcellular localization of leukemia-regulated factors including RNA-binding heterogeneous nuclear ribonucleoprotein A1 and the oncogene SET, thereby inducing reactivation of protein phosphatase 2A tumor suppressor and inhibition of BCR-ABL1 in CML-BC cells. Because XPO1 is important for leukemic cell survival, KPT-330 may represent an alternative therapy for TKI-refractory Ph(+) leukemias.

BCR-ABL1 compound mutations in tyrosine kinase inhibitor-resistant CML: frequency and clonal relationships.

BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.

PTCH1 expression at diagnosis predicts imatinib failure in chronic myeloid leukaemia patients in chronic phase.

The tyrosine kinase inhibitor (TKI) imatinib has revolutionized the management of chronic myeloid leukaemia (CML). However, around 25% of patients fail to sustain an adequate response. We sought to identify gene-expression biomarkers that could be used to predict imatinib response. The expression of 29 genes, previously implicated in CML pathogenesis, were measured by TaqMan Low Density Array in 73 CML patient samples. Patients were divided into low and high expression for each gene and imatinib failure (IF), probability of achieving CCyR, progression free survival and CML related OS were compared by Kaplan-Meier and log-rank. Results were validated in a second cohort of 56 patients, with a further technical validation using custom gene-expression assays in a conventional RT-qPCR in a sub-cohort of 37 patients. Patients with low PTCH1 expression showed a worse clinical response for all variables in all cohorts. PTCH1 was the most significant predictor in the multivariate analysis compared with Sokal, age and EUTOS. PTCH1 expression assay showed the adequate sensitivity, specificity and predictive values to predict for IF. Given the different treatments available for CML, measuring PTCH1 expression at diagnosis may help establish who will benefit best from imatinib and who is better selected for second generation TKI.

Rapidly growing mycobacteria associated with laparoscopic gastric banding, Australia, 2005-2011.

Laparoscopic gastric banding is a common bariatric procedure worldwide. Rapidly growing mycobacteria are environmental organisms increasingly seen as pathogens,often in infected prosthetic material. We report 18 cases of infection associated with laparoscopic gastric banding caused by Mycobacterium fortuitum and M. abscessus in Australia during 2005­2011. We identified cases by reviewing positive cultures at the Queensland state reference laboratory or through correspondence with clinicians, and we obtained clinical and epidemiologic data. Eleven cases of M. fortuitum and 7 cases of M. abscessus infection were identified. The port was thought to be the primary site of infection in 10 of these cases. Complications included peritonitis,band erosion, and chronic ulceration at the port site.Rapidly growing mycobacteria can infect both port and band and can occur as either an early perioperative or late infection.Combination antimicrobial therapy is used on the basis of in vitro susceptibilities. Device removal seems to be vital to successful therapy.

Responses to second-line tyrosine kinase inhibitors are durable: an intention-to-treat analysis in chronic myeloid leukemia patients.

Second-generation tyrosine kinase inhibitors (2G-TKIs) are effective at inducing complete cytogenetic responses (CCyRs) in approximately half of chronic myeloid leukemia patients treated while still in the chronic phase and after failing imatinib. It is less clear whether these responses are durable. In the present study, we report the clinical outcome of 119 patients who received a 2G-TKI as second-line treatment while still in the chronic phase. In an intention-to-treat analysis, the 4-year probabilities of overall and event-free survival were 81.9% and 35.3%, respectively. Sixty-two patients discontinued the initial 2G-TKI because of resistance or intolerance. To further explore the durability of cytogenetic responses, irrespective of the need for a third-line TKI, we used the concept of "current CCyR-survival" (c-CCyRS). The c-CCyRS at 4 years was 54.4%. After introduction of a 2G-TKI, 77 patients had a 3-month BCR-ABL1/ABL1 transcript ratio of ≤ 10% and had significantly superior overall survival (91.3% vs 72.1%, P = .02), event-free survival (49.3% vs 13.0%, P < .001), and c-CCyRS (67.2% vs 11.2%, P = .0001) compared with the 33 patients with ratios > 10%. The 3-month molecular response was the only independent predictor for overall survival. Using an intention-to-treat analysis, we have shown that the responses to second-line therapies are durable. Patients destined to fare poorly can be identified early during therapy.

Poor adherence is the main reason for loss of CCyR and imatinib failure for chronic myeloid leukemia patients on long-term therapy.

We studied the relation between adherence to imatinib measured with microelectronic monitoring systems and the probabilities of losing a complete cytogenetic response (CCyR) and of imatinib failure in 87 CCyR chronic myeloid leukemia patients receiving long-term therapy. We included in our analysis the most relevant prognostic factors described to date. On multivariate analysis, the adherence rate and having failed to achieve a major molecular response were the only independent predictors for loss of CCyR and discontinuation of imatinib therapy. The 23 patients with an adherence rate less than or equal to 85% had a higher probability of losing their CCyR at 2 years (26.8% vs 1.5%, P = .0002) and a lower probability of remaining on imatinib (64.5% vs 90.6%, P = .006) than the 64 patients with an adherence rate more than 85%. In summary, we have shown that poor adherence is the principal factor contributing to the loss of cytogenetic responses and treatment failure in patients on long-term therapy.

FISH-negative BCR::ABL1-positive e19a2 chronic myeloid leukaemia: the most cryptic of insertions.

BACKGROUND: Chronic myeloid leukaemia (CML) is one of the most well characterised human malignancies. Most patients have a cytogenetically visible translocation between chromosomes 9 and 22 which generates the pathognomonic BCR::ABL1 fusion gene. The derivative chromosome 22 ('Philadelphia' or Ph chromosome) usually harbours the fusion gene encoding a constitutively active ABL1 kinase domain. A small subset of patients have no visible translocation. Historically, these 'Philadelphia chromosome negative' patients caused diagnostic confusion between CML and other myeloproliferative neoplasms; it is now well established that the BCR::ABL1 fusion gene can be generated via submicroscopic intrachromosomal insertion of ABL1 sequence into BCR, or, more rarely, of BCR into ABL1. The fusion genes arising from cryptic insertions are not detectable via G-banded chromosome analysis [karyotype] but can nevertheless always be detected using fluorescence in situ hybridisation (FISH) and/or qualitative reverse transcriptase PCR. CASE PRESENTATION: A 43-year-old female presented with suspected CML in 2007; however, contemporaneous gold standard laboratory investigations, G-banded chromosome analysis and FISH, were both negative. The reverse transcriptase quantitative PCR (RT-qPCR) assay available at the time, which was capable of detecting the common BCR::ABL1 transcripts (e13a2/e14a2), was also negative. Upon review in 2009, the newly recommended reverse transcriptase multiplex PCR (capable of detecting all BCR::ABL1 transcripts including the atypical ones) subsequently detected an e19a2 fusion. The patient then responded to tyrosine kinase inhibitor therapy. In contrast, FISH studies of both samples with three commercially available probes remained consistently negative. Retrospective whole genome sequencing, undertaken as part of the 100,000 Genomes Project, has now revealed that the patient's BCR::ABL1 fusion gene arose via a uniquely small insertion of 122 kb ABL1 sequences into BCR. CONCLUSIONS: We present a patient with suspected chronic myeloid leukaemia whose genetic investigations were originally negative at the time of diagnosis despite the use of contemporaneous gold standard methods. This is the first report of a FISH-negative, BCR::ABL1 positive CML which demonstrates that, even after sixty years of research into one of the most well understood human malignancies, whole genome sequencing can yield novel diagnostic findings in CML.

Efficacy of tyrosine kinase inhibitors (TKIs) as third-line therapy in patients with chronic myeloid leukemia in chronic phase who have failed 2 prior lines of TKI therapy.

We analyzed a cohort of 26 patients with chronic myeloid leukemia who had failed imatinib and a second tyrosine kinase inhibitor but were still in first chronic phase and identified prognostic factors for response and outcomes. The achievement of a prior cytogenetic response on imatinib or on second-line therapy were the only independent predictors for the achievement of complete cytogenetic responses on third-line therapy. Younger age and the achievement of a cytogenetic response on second line were the only independent predictors for overall survival (OS). At 3 months, the 9 patients who had achieved a cytogenetic response had better 30-month probabilities of complete cytogenetic responses and OS than the patients who had failed to do so. Factors measurable before starting treatment with third line therapy and cytogenetic responses at 3 months can accurately predict subsequent outcome and thus guide clinical decisions.

EVI-1 oncogene expression predicts survival in chronic-phase CML patients resistant to imatinib treated with second-generation tyrosine kinase inhibitors.

Activation of the EVI-1 oncogene has been reported in acute myeloid leukemia, chronic myeloid leukemia (CML) in blast crisis, and less commonly, in chronic-phase CML patients. We screened an unselected cohort of 75 chronic-phase CML patients who had failed imatinib for expression of EVI-1 and sought a correlation with subsequent outcome on the second-generation tyrosine kinase inhibitors dasatinib (n = 61) or nilotinib (n = 14). The 8 patients (10.7%) who expressed EVI-1 transcripts detectable by real-time polymerase chain reaction had significantly lower event-free survival, progression-free survival, and overall survival than patients with undetectable transcript. The predictive value of EVI-1 expression was validated in an independent cohort. In a multivariate analysis, EVI-1 expression status and the best cytogenetic response obtained on imatinib were the only independent predictors for overall survival, progression-free survival, and event-free survival. Our data suggest that screening for EVI-1 expression at the time of imatinib failure may predict for response to second-line TKI therapy and consequently aid clinical management.