Gene expression profiling and in vitro functional studies reveal RAD54L as a potential therapeutic target in multiple myeloma.

BACKGROUND: Current advances in the molecular biology of multiple myeloma (MM) are not sufficient to fully delineate the genesis and development of this disease. OBJECTIVE: This study aimed to identify molecular targets underlying MM pathogenesis. METHODS: mRNA expression profiling for 29 samples (19 MM samples, 7 MM cell lines and 3 controls) were obtained using microarray. We evaluated the in vitro effects of RAD54L gene silencing on the proliferation, apoptosis and cell cycle distribution in KMS-28BM human MM cells using siRNA approach. Cell proliferation was determined by MTS assay while apoptosis and cell cycle distribution were analysed with flow cytometry. Gene and protein expression was evaluated using RT-qPCR and ELISA, respectively. RESULTS: Microarray results revealed a total of 5124 differentially expressed genes (DEGs), in which 2696 and 2428 genes were up-regulated and down-regulated in MM compared to the normal controls, respectively (fold change ≥ 2.0; P < 0.05). Up-regulated genes (RAD54L, DIAPH3, SHCBP1, SKA3 and ANLN) and down-regulated genes (HKDC1, RASGRF2, CYSLTR2) have never been reported in association with MM. Up-regulation of RAD54L was further verified by RT-qPCR (P < 0.001). In vitro functional studies revealed that RAD54L gene silencing significantly induced growth inhibition, apoptosis (small changes) and cell cycle arrest in G0/G1 phase in KMS-28BM (P < 0.05). Silencing of RAD54L also decreased its protein level (P < 0.05). CONCLUSIONS: This study has identified possible molecular targets underlying the pathogenesis of MM. For the first time, we reveal RAD54L as a potential therapeutic target in MM, possibly functioning in the cell cycle and checkpoint control.

Identification of FLT3 and NPM1 Mutations in Patients with Acute Myeloid Leukaemia.

Objective: The most frequent acquired molecular abnormalities and important prognostic indicators in patients with Acute Myeloid Leukaemia (AML) are fms-like tyrosine kinase-3 gene (FLT3) and nucleophosmin-1 (NPM1) mutations. Our study aims to develop a cost effective and comprehensive in-house conventional PCR method for detection of FLT3-ITD, FLT3-D835 and NPM1 mutations and to evaluate the frequency of these mutations in patients with cytogenetically normal (CN) AML in our population. Methods: A total of 199 samples from AML patients (95 women, 104 men) were included in the study. Mutation analyses were performed using polymerase chain reaction (PCR) and gene sequencing. Result: Sixty-eight patients were positive for the mutations. FLT3-ITD mutations were detected in 32 patients (16.1%), followed by FLT3-D835 in 5 (2.5%) and NPM1 in 54 (27.1%). Double mutations of NPM1 and FLT3-ITD were detected in 23 cases (11.6%). Assays validation were performed using Sanger sequencing and showed 100% concordance with in house method. Conclusion: The optimized in-house PCR assays for the detection of FLT3-ITD, FLT3-D835 and NPM1 mutations in AML patients were robust, less labour intensive and cost effective. These assays can be used as diagnostic tools for mutation detection in AML patients since identification of these mutations are important for prognostication and optimization of patient care.

Prevalence of BCR-ABL T315I Mutation in Malaysian Patients with Imatinib-Resistant Chronic Myeloid Leukemia

Objective: Chronic Myeloid Leukemia (CML) is caused by a reciprocal translocation between chromosomes 9 and 22, t(9;22) (q34;q11) which encodes for the BCR-ABL fusion protein. Discovery of Imatinib Mesylate (IM) as first line therapy has brought tremendous improvement in the management of CML. However, emergence of point mutations within the BCR-ABL gene particularly T315I mutation, affects a common BCR-ABL kinase contact residue which impairs drug binding thus contribute to treatment resistance. This study aims to investigate the BCR-ABL T315I mutation in Malaysian patients with CML. Methods: A total of 285 patients diagnosed with CML were included in this study. Mutation detection was performed using qualitative real-time PCR (qPCR). Results: Fifteen out of 285 samples (5.26%) were positive for T315I mutations after amplification with real-time PCR assay. From the total number of positive samples, six patients were in accelerated phase (AP), four in chronic phase (CP) and five in blast crisis (BC). Conclusion: Mutation testing is recommended for choosing various tyrosine kinase inhibitors (TKIs) to optimize outcomes for both cases of treatment failure or suboptimal response to imatinib. Therefore, detection of T315I mutation in CML patients are clinically useful in the selection of appropriate treatment strategies to prevent disease progression.

Whole-Exome Sequencing of ETV6/RUNX1 in Four Childhood Acute Lymphoblastic Leukaemia Cases

Background: ETV6/RUNX1 gene fusion is the most frequently seen chromosomal abnormality in childhood acute lymphobastic leukamia (ALL). However, additional genetic changes are known to be required for the development of this type of leukaemia. Therefore, we here aimed to assess the somatic mutational profile of four ALL cases carrying the ETV6/RUNX1 fusion gene using whole-exome sequencing. Methods: DNA was isolated from bone marrow samples using a QIAmp DNA Blood Mini kit and subsequently sequenced using the Illumina MiSeq system. Results: We identified 12,960 to17,601 mutations in each sample, with a total of 16,466 somatic mutations in total. Some 15,533 variants were single nucleotide polymorphisms (SNPs), 129 were substitutions, 415 were insertions and 389 were deletions. When taking into account the coding region and protein impact, 1,875 variants were synonymous and 1,956 were non-synonymous SNPs. Among non-synonymous SNPs, 1,862 were missense, 13 nonsense, 35 frameshifts, 11 nonstop, 3 misstart, 15 splices disrupt and 17 in-frame indels. A total of 86 variants were located in leukaemia-related genes of which 32 variants were located in the coding regions of GLI2, SP140, GATA2, SMAD5, KMT2C, CDH17, CDX2, FLT3, PML and MOV10L1. Conclusions: Detection and identification of secondary genetic alterations are important in identifying new therapeutic targets and developing rationally designed treatment regimens with less toxicity in ALL patients.

Detection of human papillomavirus DNA in routine cervical scraping samples: use for a national cervical cancer screening program in a developing nation.

BACKGROUND: Human papillomavirus is a well-established cause of the development of a variety of epithelial lesions in the cervix. However, as yet, incorporation of HPV testing into cervical cancer screening either as an adjunct or stand alone test is limited due to its cost. We therefore here ascertained the presence and type specificity of human papilloma virus (HPV) DNA in routine cervical scrapings. MATERIALS AND METHODS: Cervical scrapings were collected from women attending clinics for routine Pap smear screening. HPV-DNA was detected by PCR using MY09/11 and GP5+/GP6+ primer sets and genotyping was accomplished by cycle-sequencing. RESULTS: A total of 635 women were recruited into the study with mean ± SD age of 43 ± 10.5 years. Of these 92.6% (588/635) were reported as within normal limits (WNL) on cytology. The presence of HPV infection detected by nested MY/GP+-PCR was 4.4% (28/635). The overall prevalence of high-risk HPV (HR-HPV) in abnormal Pap smears was 53.8% (7/13). HPVs were also seen in 3.1% (18/588) of smears reported as WNL by cytology and 5.9% (2/34) in smears unsatisfactory for evaluation. CONCLUSIONS: The overall percentage of HPV positivity in routine cervical screening samples is comparable with abnormal findings in cytology. Conventional Pap smear 'missed' a few samples. Since HPV testing is expensive, our results may provide valuable information for strategising implementation of effective cervical cancer screening in a country with limited resources like Malaysia. If Pap smear coverage could be improved, HPV testing could be used as an adjunct method on cases with ambiguous diagnoses.

Adequacy of cellular material in split-sampling of cervical scrapings for routine cancer screening: an analysis of 702 smears.

OBJECTIVE: The aim of this study was to examine cells (split-sample) that were retained on sampling devices used to collect conventional Pap smears (primary smears) in order to evaluate specimen adequacy and cytological diagnosis of scrapings that are routinely discarded. STUDY DESIGN: Cervical scrapings from women attending routine cervical cancer screening were obtained using a cervical brush. Following primary conventional smear preparation, the same sampling devices were rinsed in Preservcyt solution (Cytyc) for subsequent monolayered thin smear (split-sample/discarded sample). The smears (conventional and ThinPrep monolayer) were examined independently by pathologists and classified using the Bethesda System. The diagnoses from discarded samples (split-sample smears) were then compared with the diagnoses made on primary conventional Pap smears. RESULTS: 702 samples were studied. Cell abnormalities was found in 14/702 conventional smear and 12/702 split-sample thin smear. The adequacy of sampling in primary smears was 94.7% compared to 88.9% in split-sample smears. Six cases of Human Papillomavirus infection was found in split-sample smear, whereas only 5 cases found in primary smear. Cohen's Kappa was 0.61 showing substantial agreement between both sampling cytological results. CONCLUSION: The cervical brush discarded after conventional smear retain adequate number of cells for diagnostic purposes.

Comparison of DR. HPV Chip Kit with hybrid capture II assay for the detection of human papillomavirus in clinical samples: a preliminary study.

Human papillomavirus (HPV) is well known as an etiological factor for the development of anogenital carcinomas. The aim of our study was to compare the performance of USFDA approved Hybrid II (HCII) Assay and recently introduced DR. HPV Chip Kit for the detection of HPV DNA in clinical cervical scrapings from 40 patients. HPV DNA testing was performed using the automated HCII Assay system and DR. HPV Chip Kit. Taking cytological results as gold standard, it was found that HCII was more sensitive (36.4%) than DR. HPV Chip Kit (18.2%) although specificity was 100% with the latter method. In addition, both these molecular methods had comparable negative and positive predictive values. It was concluded that both HCII and DR. HPV Chip Kit have comparable specificity. However, sensitivity for detection of HPV in clinical samples with HCII is almost double as compared to DR. HPV Chip Kit.