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.

Carfilzomib Enhances the Suppressive Effect of Ruxolitinib in Myelofibrosis.

As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients.

Proteasome 26S subunit, non-ATPases 1 (PSMD1) and 3 (PSMD3), play an oncogenic role in chronic myeloid leukemia by stabilizing nuclear factor-kappa B.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have revolutionized therapy for chronic myeloid leukemia (CML), paving the way for clinical development in other diseases. Despite success, targeting leukemic stem cells and overcoming drug resistance remain challenges for curative cancer therapy. To identify drivers of kinase-independent TKI resistance in CML, we performed genome-wide expression analyses on TKI-resistant versus sensitive CML cell lines, revealing a nuclear factor-kappa B (NF-κB) expression signature. Nucleocytoplasmic fractionation and luciferase reporter assays confirmed increased NF-κB activity in the nucleus of TKI-resistant versus sensitive CML cell lines and CD34+ patient samples. Two genes that were upregulated in TKI-resistant CML cells were proteasome 26S subunit, non-ATPases 1 (PSMD1) and 3 (PSMD3), both members of the 19S regulatory complex in the 26S proteasome. PSMD1 and PSMD3 were also identified as survival-critical genes in a published small hairpin RNA library screen of TKI resistance. We observed markedly higher levels of PSMD1 and PSMD3 mRNA in CML patients who had progressed to the blast phase compared with the chronic phase of the disease. Knockdown of PSMD1 or PSMD3 protein correlated with reduced survival and increased apoptosis in CML cells, but not in normal cord blood CD34+ progenitors. Luciferase reporter assays and immunoblot analyses demonstrated that PSMD1 and PSMD3 promote NF-κB protein expression in CML, and that signal transducer and activator of transcription 3 (STAT3) further activates NF-κB in scenarios of TKI resistance. Our data identify NF-κB as a transcriptional driver in TKI resistance, and implicate PSMD1 and PSMD3 as plausible therapeutic targets worthy of future investigation in CML and possibly other malignancies.

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.

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy.

Molecular Monitoring of Chronic Myeloid Leukemia.

Molecular diagnosis and measurement of minimal residual disease (MRD) in patients with chronic myeloid leukemia (CML) is essential for clinical management. In the era of tyrosine kinase inhibitor therapy molecular tests including BCR-ABL1 transcript monitoring and kinase domain mutation analysis are the main tools used to inform choice of treatment, appropriate dosage and even whether therapy can be safely withdrawn. Quantitation of BCR-ABL1 oncogene transcript by real-time quantitative PCR (qPCR) is currently the gold-standard method for monitoring as it provides superior sensitivity over karyotyping and fluorescent in situ hybridization (FISH). Here we describe step-by-step methods of RNA conversion to cDNA along with the qPCR protocol which is used in one of the main reference laboratories for this test.

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.

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.

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.

Sexual orientation and medical history among Iranian people with Complete Androgen Insensitivity Syndrome and Congenital Adrenal Hyperplasia.

OBJECTIVE: To report sexual orientation, relationship status and medical history of Iranian people with Differences of Sex Development (DSD) who were raised female. METHODS: Our participants consisted of nineteen 46,XY individuals with Complete Androgen Insensitivity Syndrome (CAIS) and eighteen 46,XX individuals with Congenital Adrenal Hyperplasia (CAH) who were raised as females and older than 13years. As well as their relationship status and detailed medical history, an expert psychiatrist assessed their sexual orientation by a semi-structured psychiatric interview with them and, where applicable, their parents. RESULTS: Five percent of CAH participants and 42% of CAIS participants were in a relationship, which was significantly different. All CAH individuals had been diagnosed at birth; 89% of CAIS had been diagnosed after puberty and due to primary amenorrhea and 11% were diagnosed in childhood due to inguinal hernia. Genital reconstructive surgery had been performed in 100% of CAH participants and 37% of CAIS. Regarding sexual contact experiences and sexual fantasies (androphilic, gynephilic or both), no significant differences were found. However, CAH females had significantly more gynephilic dreams (P=0.045). CONCLUSION: This study, notable as one of the rare from a non-western culture, described sexual, medical and socioeconomic status of 46,XX CAH and 46,XY CAIS individuals living in Iran. Although broadly in line with previous findings from Western cultures, Iranian CAH individuals had fewer romantic relationships, but in contrast to previous studies their sexual orientation was only different from CAIS in the contents of sexual dreams.

Next-Generation Sequencing-Assisted DNA-Based Digital PCR for a Personalized Approach to the Detection and Quantification of Residual Disease in Chronic Myeloid Leukemia Patients.

Recent studies indicate that 40% of chronic myeloid leukemia patients who achieve sustained undetectable BCR-ABL1 transcripts on tyrosine kinase inhibitor therapy remain disease-free after drug discontinuation. In contrast, 60% experience return of detectable disease and have to restart treatment, thus highlighting the need for an improved method of identifying patients with the lowest likelihood of relapse. Here we describe the validation of a personalized DNA-based digital PCR (dPCR) approach for quantifying very low levels of residual disease, which involves the rapid identification of t(9;22) fusion junctions using targeted next-generation sequencing coupled with the use of a dPCR platform. t(9;22) genomic breakpoints were successfully mapped in samples from 32 of 32 patients with early stage disease. Disease quantification by DNA-based dPCR was performed using the Fluidigm BioMark platform on 46 follow-up samples from 6 of the 32 patients, including 36 samples that were in deep molecular remission. dPCR detected persistent disease in 81% of molecular-remission samples, outperforming both RT-dPCR (25%) and DNA-based quantitative PCR (19%). We conclude that dPCR for BCR-ABL1 DNA is the most sensitive available method of residual-disease detection in chronic myeloid leukemia and may prove useful in the management of tyrosine kinase inhibitor withdrawal.

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.

BCL3 rearrangement, amplification and expression in diffuse large B-cell lymphoma.

AIMS: Aim of the study is to investigate diffuse large B-cell lymphoma (DLBCL) for the presence of BCL3 gene rearrangement and protein expression and to correlate these with immunophenotypic subsets of DLBCL. We aimed to investigate the pathogenetic implication of BCL3 in DLBCL. METHODS AND RESULTS: Tissue microarray sections from 78 DLBCLs were evaluated for BCL3 protein expression using immunohistochemistry and for BCL3 and IGH rearrangement using Fluorescent in situ hybridisation (FISH) with split-apart probes. BCL3 expression was positive in 36/78 cases, of which BCL3 rearrangement was seen seen in one case. Three additional cases showed evidence of trisomy of BCL3/chromosome 19, and two of these three cases showed BCL3 expression. The four cases with FISH-detectable abnormalities showed MUM1 expression and had a non-germinal center (GC) phenotype. The median [and inter-quartile range (IQR)] percentage of BCL3-positive cells in MUM1-positive and MUM1-negative subsets was 65% (5-85%) and 5% (0-20%), respectively (P < 0.001). The median (IQR) percentage of BCL3-positive cells among GC and non-GC subsets of DLBCLs was 12% (12-81%) and 60% (6-87%), respectively (P = 0.022). CONCLUSION: Rearrangement or amplification involving the BCL3 gene is a rare event in DLBCL but is likely to play a role in the pathogenesis of a minority of de novo DLBCL. BCL3 over-expression is more frequent and occurs in the absence of rearrangement or amplification and is a feature of the non-GC subset of DLBCL.

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.

T cell lymphoblastic leukaemia/lymphoma associated with a microenvironment of thymic asteroid B cells in the bone marrow.

AIMS: Asteroid B cells are a component of normal thymus. It is currently unclear whether these cells are identifiable in T cell lymphoblastic leukaemia/lymphoma (T-ALL/LBL) of the thymus. The aim of this study was to identify asteroid B cells both in thymic and extrathymic tissue involved by T-ALL/LBL. METHODS AND RESULTS: Thymic, lymph node (LN) and bone marrow trephine biopsy (BMTB) samples from eight patients with T-ALL/LBL were reviewed. All had been investigated by immunohistochemistry and one by fluorescent in situ hybridization (FISH). The BMTB samples of two of eight T-ALL/LBLs and LN sample in one of them showed the presence of asteroid-shaped B cells with dendritic cytoplasmic processes. These B cells also expressed CD23 and the features were akin to the unique thymic asteroid B cells. Both patients had aggressive/resistant disease. Cytogenetic analysis in one showed a complex translocation involving the T cell receptor beta (TCRB) gene at 7q35 and a distal region of 9q known to harbour the NOTCH1 gene. CONCLUSION: This is the first report of T-ALL/LBL documenting the presence of an asteroid B cell-rich microenvironment at bone marrow and LN sites. In this small subset, T-ALL/LBL cells are possibly dependent upon asteroid B cells, and whether targeting of asteroid B cells with anti-CD20 monoclonal antibody in such cases will result in clinical benefit remains to be determined.

Specific patterns of chromosomal gains and losses associate with t(3;14), t(8;14), and t(14;18) in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease. Certain chromosomal translocations are associated with clinical outcome, but it is likely that there are both tumor suppressor genes and oncogenes that cooperate with the primary translocations. We have used the Mitelman database to compare chromosomal losses and gains of DLBCL possessing t(14;18), t(8;14), or t(3;14) with DLBCL lacking any of these translocations. The data we obtained are low resolution, but results for t(3;14) validate the methodology. In accord with the literature, loss of 6q was associated with t(3;14). Chromosomes 11, 13, and X were gained significantly in t(3;14), whereas 8p23 was lost. Cases with t(14;18) were associated with gains of chromosomes 7 and 12; cases with t(8;14) were associated with gains of chromosomes 1 and 4.

Does a rise in the BCR-ABL1 transcript level identify chronic phase CML patients responding to imatinib who have a high risk of cytogenetic relapse?

BCR-ABL1 transcript numbers were monitored in 161 patients who started treatment with imatinib early after diagnosis of chronic myeloid leukaemia in chronic phase and achieved complete cytogenetic responses (CCyR). A confirmed doubling in BCR-ABL1/ABL1 transcript levels was found to be a significant factor for predicting loss of CCyR [relative risk (RR) 8.3, P < 0.0001] and progression to advanced phase (RR 0.07, P = 0.03) provided that the eventual BCR-ABL1/ABL1 transcript level exceeded 0.05%; increases that never exceeded 0.05% had no predictive value. The finding of a kinase domain mutation in a patient in CCyR, though rare, also predicted for loss of CCyR.

Phenotype of blasts in chronic myeloid leukemia in blastic phase-Analysis of bone marrow trephine biopsies and correlation with cytogenetics.

We identified different phenotypic subsets among 62 cases of chronic myeloid leukemia (CML) in blast crisis (BC) (26% B-lymphoblastic, and 74% various myeloblastic subsets) on bone marrow trephines and correlated the blast-phenotype with cytogenetics. Five of myeloid-BC had an associated 3q26 abnormality and two of these showed a megakaryoblastic-phenotype. While myeloid-BC was associated with additional copies of Philadelphia (Ph) (29%) (p=0.08), numerical abnormalities (51%) (p=0.007), trisomy-8 (29%) (p=0.08) and 17p-loss (22%), none of lymphoid-BC showed these abnormalities. Among myeloid-BC, CD34-negative cases were more often associated with trisomy-8, 17p-loss and numerical abnormalities, and the CD117-negative subset with additional copies of Ph (p<0.05).