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Clinical outcome and mutations of 96 core-binding factor acute myeloid leukemia (AML) patients 18-60 years old were examined. Complete remission (CR) after induction was 94.6%. There was no significant difference in CR, leukemia-free-survival (LFS) and overall survival (OS) between t(8;21) (N=67) and inv(16) patients (N=29). Univariate analysis showed hematopoietic stem cell transplantation at CR1 as the only clinical parameter associated with superior LFS. Next-generation sequencing based on a myeloid gene panel was performed in 72 patients. Mutations in genes involved in cell signaling were associated with inferior LFS and OS, whereas those in genes involved in DNA methylation were associated with inferior LFS. KIT activation loop (AL) mutations occurred in 25 patients, and were associated with inferior LFS (P=0.003) and OS (P=0.001). TET2 mutations occurred in 8 patients, and were associated with significantly shorter LFS (P=0.015) but not OS. Patients negative for KIT-AL and TET2 mutations (N=41) had significantly better LFS (P<0.001) and OS (P=0.012) than those positive for both or either mutation. Multivariate analysis showed that KIT-AL and TET2 mutations were associated with inferior LFS, whereas age ⩾40 years and marrow blast ⩾70% were associated with inferior OS. These observations provide new insights that may guide better treatment for this AML subtype.

Background: Genomic techniques in recent years have allowed the identification of many mutated genes important in the pathogenesis of acute myeloid leukemia (AML). Together with cytogenetic aberrations, these gene mutations are powerful prognostic markers in AML and can be used to guide patient management, for example selection of optimal post-remission therapy. The mutated genes also hold promise as therapeutic targets themselves. We evaluated the applicability of a gene panel for the detection of AML mutations in a diagnostic molecular pathology laboratory., Methods: Fifty patient samples comprising 46 AML and 4 other myeloid neoplasms were accrued for the study. They consisted of 19 males and 31 females at a median age of 60 years (range: 18-88 years). A total of 54 genes (full coding exons of 15 genes and exonic hotspots of 39 genes) were targeted by 568 amplicons that ranged from 225 to 275 bp. The combined coverage was 141 kb in sequence length. Amplicon libraries were prepared by TruSight myeloid sequencing panel (Illumina, CA) and paired-end sequencing runs were performed on a MiSeq (Illumina) genome sequencer. Sequences obtained were analyzed by in-house bioinformatics pipeline, namely BWA-MEM, Samtools, GATK, Pindel, Ensembl Variant Effect Predictor and a novel algorithm ITDseek., Results: The mean count of sequencing reads obtained per sample was 3.81 million and the mean sequencing depth was over 3000X. Seventy-seven mutations in 24 genes were detected in 37 of 50 samples (74 %). On average, 2 mutations (range 1-5) were detected per positive sample. TP53 gene mutations were found in 3 out of 4 patients with complex and unfavorable cytogenetics. Comparing NGS results with that of conventional molecular testing showed a concordance rate of 95.5 %. After further resolution and application of a novel bioinformatics algorithm ITDseek to aid the detection of FLT3 internal tandem duplication (ITD), the concordance rate was revised to 98.2 %., Conclusions: Gene panel testing by NGS approach was applicable for sensitive and accurate detection of actionable AML gene mutations in the clinical laboratory to individualize patient management. A novel algorithm ITDseek was presented that improved the detection of FLT3-ITD of varying length, position and at low allelic burden.

Approximately 5%-10% of breast cancers are due to genetic predisposition caused by germline mutations; the most commonly tested genes are BRCA1 and BRCA2 mutations. Some mutations are unique to one family and others are recurrent; the spectrum of BRCA1/BRCA2 mutations varies depending on the geographical origins, populations or ethnic groups. In this review, we compiled data from 11 participating Asian countries (Bangladesh, Mainland China, Hong Kong SAR, Indonesia, Japan, Korea, Malaysia, Philippines, Singapore, Thailand and Vietnam), and from ethnic Asians residing in Canada and the USA. We have additionally conducted a literature review to include other Asian countries mainly in Central and Western Asia. We present the current pathogenic mutation spectrum of BRCA1/BRCA2 genes in patients with breast cancer in various Asian populations. Understanding BRCA1/BRCA2 mutations in Asians will help provide better risk assessment and clinical management of breast cancer., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background: Large tumor suppressor 2 (LATS2) gene is a putative tumor suppressor gene with potential roles in regulation of cell proliferation and apoptosis in lung cancer. The aim of this study is to explore the association of aberrant LATS2 expression with EGFR mutation and survival in lung adenocarcinoma (AD), and the effects of LATS2 silencing in both lung AD cell lines., Methods: LATS2 mRNA and protein expression in resected lung AD were correlated with demographic characteristics, EGFR mutation and survival. LATS2-specific siRNA was transfected into four EGFR wild-type (WT) and three EGFR mutant AD cell lines and the changes in LATS2 expression and relevant signaling molecules before and after LATS2 knockdown were assayed., Results: Fifty resected lung AD were included (M:F=23:27, smokers:non-smokers=19:31, EGFR mutant:wild-type=21:29) with LATS2 mRNA levels showed no significant difference between gender, age, smoking and pathological stages while LATS2 immunohistochemical staining on an independent set of 79 lung AD showed similar trend. LATS2 mRNA level was found to be a significant independent predictor for survival status (disease-free survival RR=0.217; p=0.003; Overall survival RR=0.238; p=0.036). siRNA-mediated suppression of LATS2 expression resulted in augmentation of ERK phosphorylation in EGFR wild-type AD cell lines with high basal LATS2 expression, discriminatory modulation of Akt signaling between EGFR wild-type and mutant cells, and induction of p53 accumulation in AD cell lines with low baseline p53 levels., Conclusions: LATS2 expression level is predictive of survival in patients with resected lung AD. LATS2 may modulate and contribute to tumor growth via different signaling pathways in EGFR mutant and wild-type tumors., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Activating mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer predict for a favorable clinical response to tyrosine kinase inhibitor therapy. Although Sanger sequencing is a conventional method to detect EGFR gene mutations, multiplex real-time allele-specific polymerase chain reaction (PCR) systems are increasingly used in the routine molecular diagnostic setting. We aim to evaluate 2 proprietary real-time PCR assays (cobas and therascreen) against Sanger sequencing in the detection of EGFR gene mutations. The overall concordance rate between cobas and therascreen assays with Sanger sequencing was 89% and 88%, respectively, and increased to 96% and 98%, respectively, if the mutations not covered were excluded. The cobas assay showed a superior coverage of exon 20 mutations, but L861Q was not targeted. The nature of specimen, DNA integrity, and tumor cell content are factors that affect the assay performance. DNA extracted from cell block and clot of pleural fluid gave rise to 1 invalid call and 1 false-negative result by the cobas assay and 1 missed T790M mutation and 1 false-negative result by the therascreen assay. Both assays are around 5 times more expensive compared with Sanger sequencing in terms of reagent cost. We conclude that both assays prove to be a rapid, simple, and validated method in detecting the most common and clinically significant EGFR gene mutations in non-small cell lung cancer. Although less convenient compared with real-time PCR assays, Sanger sequencing is cheaper in terms of reagent cost and allows the detection of rare or novel EGFR gene mutations.

Background: This study investigated whether there were differential survival outcomes to first-line tyrosine kinase inhibitors (TKI) in patients with metastatic non-small-cell lung cancer harboring different subtypes of exon 19 and exon 21 mutations on epidermal growth factor receptor (EGFR)., Methods: Of 452 patients with stage IIIB and IV non-small-cell lung cancer, 192 patients (42.5%) harbored EGFR mutation and 170 (37.5%) received TKI as first-line treatment. EGFR mutation analysis was performed by direct sequencing. Survival and response outcome were compared among different subtypes of exon 19 and exon 21 EGFR mutations in these 170 patients., Results: Patients harboring exon 19 18-nucleotide deletion (delL747&#95;P753insS) had the shortest median progression-free survival (PFS) (6.5 months), followed by those with 15-nucleotide deletion (delE746&#95;A750) (12.4 months) and mixed insertion/substitution mutations (22.3 months; p = 0.012). However, patients who had exon 19 deletions starting on codon E746 had better median PFS (14.2 months) than those starting on L747 (6.5 months; hazard ratio, 0.445; 95% confidence interval [0.219-0.903]; p = 0.021). Besides, exon 21 L858R derived a longer median PFS than L861R/L861Q (11.4 months versus 2.1 months, respectively; hazard ratio, 0.298; 95% confidence interval [0.090-0.980]; p = 0.034)., Conclusions: Different subtypes of EGFR exon 19 and 21 mutations exhibited differential survival to first-line TKI therapy. Detailed sequence evaluation of exon 19 deletions may provide important prognostic information on survival outcome after TKI.

Activating mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer predicts a significantly higher clinical response rate to tyrosine kinase inhibitors targeting EGFR, and it is currently recommended that patients under consideration for EGFR TKI as first-line therapy be tested for such mutations to determine the appropriateness of treatment. For lung cancer patients who present with advanced stage disease where surgical treatment is not indicated, cytology specimens obtained through bronchoscopy, drainage of body fluid, or fine-needle aspiration are the only means to obtain tumor cells for tissue diagnosis and EGFR gene mutation testing. We reviewed the experience of 1,410 consecutive EGFR mutation testing requests at a single institution in Hong Kong that comprised 269 cytology specimens and 1,141 surgical specimens. The material inadequacy issue in cytology specimens may be overcome by tumor cell enrichment strategies and employment of mutation detection techniques with increased analytical sensitivity. The use of cytology specimens to test for predictive molecular cancer biomarkers is without a doubt expected to increase, and cytopathologists should be closely engaged with the clinicians in the therapeutic process and become acquainted with new technology in order to directly participate in personalized oncology care., (Copyright © 2012 S. Karger AG, Basel.)

Background: Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China., Methodology/principal Findings: A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM) analysis. Among the 451 probands analyzed, 69 (15.3%) deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations (c.470&#95;471delCT, c.3342&#95;3345delAGAA, c.5406+1&#95;5406+3delGTA and c.981&#95;982delAT) accounted for 34.5% (10/29) of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations (c.2808&#95;2811delACAA, c.3109C>T, c.7436&#95;7805del370 and c.9097&#95;9098insA) accounted for 40% (16/40) of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated., Conclusion: In this study, our findings suggest that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.

Germline mutations in the two breast cancer susceptibility genes, BRCA1 and BRCA2 account for a significant portion of hereditary breast/ovarian cancer. De novo mutations such as multiple exon deletion are rarely occurred in BRCA1 and BRCA2. During our mutation screening for BRCA1/2 genes to Chinese women with risk factors for hereditary breast/ovarian cancer, we identified a novel germline mutation, consisting of a deletion from exons 1 to 12 in BRCA1 gene, in a patient diagnosed with early onset triple negative breast cancer with no family history of cancer. None of her parents carried the mutation and molecular analysis showed that this novel de novo germline mutation resulted in down-regulation of BRCA1 gene expression.

We describe a 75-year-old Chinese man who presented with three separate tumors in three different lobes of the lung, without evidence of mediastinal or systemic involvement. All three tumors were surgically resected by minimal invasive approach. Based on a differing epidermal growth factor receptor (EGFR) mutation status, the tumors were characterized as synchronous triple primary rather than intrapulmonary metastases. This report highlights the clinical usefulness of molecular cancer biomarkers to determine prognosis and to guide management decision in multiple lung tumors., (2010 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Aims: Mutation of the KRAS gene predicts the clinical response to the monoclonal antibody cetuximab in patients with advanced colorectal cancer (CRC). This study aimed to perform KRAS mutation detection on formalin-fixed paraffin-embedded (FFPE) tumour tissue by two different methods for comparison., Methods: The FFPE sample was microdissected to enrich for tumour cells. KRAS exon 2 mutations were performed on 100 Chinese patients with CRC by direct nucleotide sequencing and high-resolution melting (HRM) analysis., Results: KRAS exon 2 mutations were detected in a total of 62 patients with the two methods combined, comprising 11 different mutant alleles. Three common mutations p.Gly12Asp, p.Gly12Val and p.Gly13Asp accounted for approximately 70% of all cases. The concordant rate between the two methods was 95%. Four mutations not initially detected by direct sequencing were identified by HRM and confirmed by sequencing of the HRM amplicons. One mutation detected by direct sequencing was inadvertently grouped as a wild-type allele by HRM software, but this was readily rectified through manual review., Conclusion: HRM analysis is a sensitive method of detecting KRAS mutation on FFPE tumour tissue to guide cetuximab treatment and is applicable to routine molecular diagnostic service. Utilisation of HRM to screen for mutations upfront economises the resource used in the sequencing reaction.

An extended family with three individuals affected by two different forms of double heterozygosity for beta-thalassemia and Hb New York is reported. Double heterozygosity of Hb New York [beta 113 GTG-->GAG; VAL-->GLU] and beta degrees codon 17 was detected in a fetus following prenatal screening for thalassemia. The father and a paternal aunt were also found to be heterozygous for Hb New York and beta degrees IVSII-654. Both adults had clinical and hematological features consistent with beta-thalassemia trait. The affected child was followed up after birth and manifested the typical course of a thalassemia trait, with no signs of organomegaly or overt hemolysis. Observations strongly suggest that double heterozygosity of Hb New York and beta degrees thalassemia has mild, if any, clinical symptoms, and is not an indication of therapeutic abortion when detected antenatally.

Introduction: Germline mutations of BRCA1 and BRCA2 account for the majority of hereditary breast cancers, many of which are classified as variants of unknown significance (VUS). We report the identification of a novel BRCA2 variant (c.7806-9T > G) in a Chinese family with multiple breast cancers and document it as a pathogenic mutation., Methods: The proband in this family was diagnosed with breast cancer at age 50 with a strong family history of breast cancer. DNA and RNA were extracted from the blood of the proband and her family, and was used for BRCA gene mutation/deletion screening and RNA splicing analysis., Results: BRCA2 c.7806-9T > G was identified in the proband, which was suggestive of a variant. This change was also found in two sisters of the proband with a history of breast cancer, as well as from the proband's maternal gastric cancer. The only sibling free of breast cancer did not carry the BRCA2 variant, thus demonstrating that the mutation segregates with the clinical phenotype in this family. RNA analysis on the proband blood sample revealed three aberrant splicing variants: c.7806&#95;7874del, c.7806&#95;7976del, and c.7806-8&#95;7806-1ins. The latter causes a frameshift and creates a truncated protein, whilst the other two splicing variants resulted in shorter forms of the protein., Conclusions: The identified BRCA2 c.7806-9T > G [Genbank: DQ889340] was found to be pathogenic, based on aberrant splicing events resulting in the formation of truncated protein products. Thus, better understanding and classification of BRCA variants as neutral or disease causing has important implications for genetic counseling so that appropriate management can be given.

FLT-3 aberrations that occur as an internal tandem duplication (ITD) or a mutation at the activation-loop position 835, D835, are common in acute promyelocytic leukaemia (APL). We investigated the clinicopathological associations and prognostic impact of FLT-3 aberrations in a cohort of APL patients. FLT-3 exons 11 and 12 were amplified by polymerase chain reaction (PCR), and the ITD was recognized as an increase in the size of the PCR product. FLT-3 exon 17 was amplified, and D835 mutation was identified by loss of an EcoRV site, followed by DNA sequencing. Of 82 patients studied, FLT-3 aberrations were detected in 35 cases (43%) at diagnosis (ITD: 16; D835 mutation: 18; ITD + D835 mutation: 1). FLT-3 ITD, but not D835 mutations, was significantly associated with higher presentation white blood cell count (WBC) and microgranular morphology. Early/induction deaths were related to male sex and high presentation WBC. There was a trend for FLT-3 ITD to be associated with non-remission (P = 0.06). For disease-free survival, high WBC was the only significant adverse factor. Male sex, high WBC and FLT-3 ITD were significant adverse factors for overall survival. These findings have important implications on the possible use of FLT-3 inhibitors in the treatment of APL.