Identification of germline alterations in breast cancer predisposition genes among Malaysian breast cancer patients using panel testing.

Although an association between protein-truncating variants and breast cancer risk has been established for 11 genes, only alterations in BRCA1, BRCA2, TP53 and PALB2 have been reported in Asian populations. Given that the age of onset of breast cancer is lower in Asians, it is estimated that inherited predisposition to breast cancer may be more significant. To determine the potential utility of panel testing, we investigated the prevalence of germline alterations in 11 established and 4 likely breast cancer genes in a cross-sectional hospital-based cohort of 108 moderate to high-risk breast cancer patients using targeted next generation sequencing. Twenty patients (19%) were identified to carry deleterious mutations, of whom 13 (12%) were in the BRCA1 or BRCA2, 6 (6%) were in five other known breast cancer predisposition genes and 1 patient had a mutation in both BRCA2 and BARD1. Our study shows that BRCA1 and BRCA2 account for the majority of genetic predisposition to breast cancer in our cohort of Asian women. Although mutations in other known breast cancer genes are found, the functional significance and breast cancer risk have not yet been determined, thus limiting the clinical utility of panel testing in Asian populations.

Frequent occurrence of gastric cancer in Asian kindreds with Li-Fraumeni syndrome.

Type of cancer and age of onset in individuals with inherited aberrations in the tumour suppressor gene TP53 are variable, possibly influenced by genetic modifiers and different environmental exposure. Since 2009, the modified Chompret criteria (MCC) have been used to identify individuals for TP53 mutation screening. Using the TP53 mutation database maintained by the International Agency for Research on Cancer (IARC), we investigated if the MCC, mainly developed for a Caucasian population, was also applicable in Asia. We identified several differences in Asian families compared with similar Caucasian cohorts, suggesting that identification and management of Li-Fraumeni syndrome in Asia do not completely mirror that of North America and Western Europe. Early gastric cancer (<40 years) may be considered a new addition to the MCC especially for Asian families.

FGF receptor genes and breast cancer susceptibility: results from the Breast Cancer Association Consortium.

BACKGROUND: Breast cancer is one of the most common malignancies in women. Genome-wide association studies have identified FGFR2 as a breast cancer susceptibility gene. Common variation in other fibroblast growth factor (FGF) receptors might also modify risk. We tested this hypothesis by studying genotyped single-nucleotide polymorphisms (SNPs) and imputed SNPs in FGFR1, FGFR3, FGFR4 and FGFRL1 in the Breast Cancer Association Consortium. METHODS: Data were combined from 49 studies, including 53 835 cases and 50 156 controls, of which 89 050 (46 450 cases and 42 600 controls) were of European ancestry, 12 893 (6269 cases and 6624 controls) of Asian and 2048 (1116 cases and 932 controls) of African ancestry. Associations with risk of breast cancer, overall and by disease sub-type, were assessed using unconditional logistic regression. RESULTS: Little evidence of association with breast cancer risk was observed for SNPs in the FGF receptor genes. The strongest evidence in European women was for rs743682 in FGFR3; the estimated per-allele odds ratio was 1.05 (95% confidence interval=1.02-1.09, P=0.0020), which is substantially lower than that observed for SNPs in FGFR2. CONCLUSION: Our results suggest that common variants in the other FGF receptors are not associated with risk of breast cancer to the degree observed for FGFR2.

A review of breast cancer research in malaysia.

Four hundred and nineteen articles related to breast cancer were found in a search through a database dedicated to indexing all original data relevant to medicine published in Malaysia between the years 2000-2013. One hundred and fifty four articles were selected and reviewed on the basis of clinical relevance and future research implications. Overall, Malaysian women have poor survival from breast cancer and it is estimated that half of the deaths due to breast cancer could be prevented. Five-year survival in Malaysia was low and varies among different institutions even within the same disease stage, suggesting an inequity of access to optimal treatment or a lack of compliance to optimal treatment. Malaysian women have poor knowledge of the risk factors, symptoms and methods for early detection of breast cancer, leading to late presentation. Moreover, Malaysian women experience cancer fatalism, belief in alternative medicine, and lack of autonomy in decision making resulting in delays in seeking or avoidance of evidence-based medicine. There are ethnic differences in estrogen receptor status, HER2 overexpression and incidence of triple negative breast cancer which warrant further investigation. Malay women present with larger tumours and at later stages, and even after adjustment for these and other prognostic factors (stage, pathology and treatment), Malay women have a poorer survival. Although the factors responsible for these ethnic differences have not been elucidated, it is thought that pharmacogenomics, lifestyle factors (such as weight-gain, diet and exercise), and psychosocial factors (such as acceptance of 2nd or 3rd line chemotherapy) may be responsible for the difference in survival. Notably, survivorship studies show self-management programmes and exercise improve quality of life, highlighting the need to evaluate the psychosocial impact of breast cancer on Malaysian women, and to design culturally-, religiously- and linguistically-appropriate psycho-education programmes to help women cope with the disease and improve their quality of life. Research done in the Caucasian populations may not necessarily apply to local settings and it is important to embark on local studies particularly prevention, screening, diagnostic, prognostic, therapeutic and psychosocial research.

Targeted prostate cancer screening in men with mutations in BRCA1 and BRCA2 detects aggressive prostate cancer: preliminary analysis of the results of the IMPACT study.

OBJECTIVE: To evaluate the role of targeted prostate cancer screening in men with BRCA1 or BRCA2 mutations, an international study, IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls), was established. This is the first multicentre screening study targeted at men with a known genetic predisposition to prostate cancer. A preliminary analysis of the data is reported. PATIENTS AND METHODS: Men aged 40-69 years from families with BRCA1 or BRCA2 mutations were offered annual prostate specific antigen (PSA) testing, and those with PSA > 3 ng/mL, were offered a prostate biopsy. Controls were men age-matched (± 5 years) who were negative for the familial mutation. RESULTS: In total, 300 men were recruited (205 mutation carriers; 89 BRCA1, 116 BRCA2 and 95 controls) over 33 months. At the baseline screen (year 1), 7.0% (21/300) underwent a prostate biopsy. Prostate cancer was diagnosed in ten individuals, a prevalence of 3.3%. The positive predictive value of PSA screening in this cohort was 47·6% (10/21). One prostate cancer was diagnosed at year 2. Of the 11 prostate cancers diagnosed, nine were in mutation carriers, two in controls, and eight were clinically significant. CONCLUSIONS: The present study shows that the positive predictive value of PSA screening in BRCA mutation carriers is high and that screening detects clinically significant prostate cancer. These results support the rationale for continued screening in such men.

A retrospective review of uterine scar pregnancies: single centre experience.

PURPOSE: Our aim was to present our experience with the management of six women with uterine scar pregnancies in KK Women's and Children's Hospital, Singapore. METHODS: The medical records of women with a pregnancy in previous uterine scar that had been diagnosed in our department during 2004-2008 were reviewed. RESULTS: Out of six women, one woman presented in mid-trimester, at 16 weeks with severe abdominal pain and persistent vomiting. She underwent a hysterectomy complicated with massive haemorrhage. The other five women presented in first trimester. Two women had excision of the scar with the sac, two had ultrasound-guided injection of methotrexate in the sac and one had systemic methotrexate. In all cases, maternal recovery was complete. Uterine scar pregnancy was diagnosed by ultrasonography. CONCLUSION: Women at a risk appear to be those with multiple Caesarean sections, termination of pregnancy and myomectomy. Operative as well as medical treatments have been reported for scar pregnancy. Surgical treatment includes excision of trophoblastic tissues by laparotomy or laparoscopy whilst medical treatment includes local and/or systemically administered methotrexate. Although many interventions have been described, optimal treatment is still not known and they remain a challenge.

Elevated plasma and synovial fluid interleukin-8 and interleukin-18 may be associated with the pathogenesis of knee osteoarthritis.

PURPOSE: Osteoarthritis (OA) of the knee is a multifactorial degenerative disease typically defined as the 'wear and tear' of articular joint cartilage. However, recent studies suggest that OA is a disease arising from chronic low-grade inflammation. We conducted a study to investigate the relationship between chronic inflammatory mediators present in both the systemic peripheral blood system and localised inflammation in synovial fluid (SF) of OA and non-OA knees; and subsequently made direct comparative analyses to understand the mechanisms that may underpin the processes involved in OA. METHODS: 20-Plex proteins were quantified using Human Magnetic Luminex® assay (R&D Systems, USA) from plasma and SF of OA (n = 14) and non-OA (n = 14) patients. Ingenuity Pathway Analysis (IPA) software was used to predict the relationship and possible interaction of molecules pertaining to OA. RESULTS: There were significant differences in plasma level for matrix metalloproteinase (MMP)-3, interleukin (IL)-27, IL-8, IL-4, tumour necrosis factor-alpha, MMP-1, IL-15, IL-21, IL-10, and IL-1 beta between the groups, as well as significant differences in SF level for IL-15, IL-8, vascular endothelial growth factor (VEGF), MMP-1, and IL-18. Our predictive OA model demonstrated that toll-like receptor (TLR) 2, macrophage migration inhibitory factor (MIF), TLR4 and IL-1 were the main regulators of IL-1B, IL-4, IL-8, IL-10, IL-15, IL-21, IL-27, MMP-1 and MMP-3 in the plasma system; whilst IL-1B, TLR4, IL-1, and basigin (BSG) were the regulators of IL-4, IL-8, IL-10, IL-15, IL-18, IL-21, IL-27, MMP-1, and MMP-3 in the SF system. CONCLUSION: The elevated plasma IL-8 and SF IL-18 may be associated with the pathogenesis of OA via the activation of MMP-3.

Clinical and pathologic differences between BRCA1-, BRCA2-, and non-BRCA-associated breast cancers in a multiracial developing country.

BACKGROUND: Mutations in BRCA1 and BRCA2 confer an increased risk to breast and other cancers, but to date there have only been limited numbers of studies of BRCA1- and BRCA2-associated cancers among Asians. Malaysia is a multiracial country with three main races: Malays, Chinese, Indians. We determined whether tumor pathologic features and clinical features differ in patients with and without BRCA mutations in this Asian population. METHODS: We conducted a retrospective review of the medical records of 152 women with breast cancer who underwent genetic testing for BRCA mutations. The patients self-reported ethnicity, age at onset, and clinical stage at diagnosis and tumor pathology were reviewed. RESULTS: A total of 31 patients carried germline deleterious mutations (16 BRCA1, 15 BRCA2). We found that tumors in BRCA1 carriers were more likely to be estrogen receptor (ER)-negative and progesterone receptor (PR)-negative. HER2 was more likely to be negative in both BRCA1 and BRCA2 subjects compared with non-BRCA subjects. We found a strong association between triple-negative status and BRCA1 carriers. In addition, tumors in BRCA1 carriers were more likely to be higher grade than those in BRCA2 and non-BRCA carriers; but the difference was not statistically significant. CONCLUSIONS: These results suggest that tumors associated with BRCA1 mutations are distinct from those of BRCA2-associated and non-BRCA-associated breast cancers, and that the tumors associated with BRCA2 mutations are similar to the non-BRCA-associated breast cancers. Further studies are required to determine if the prognosis is different in each of these groups and the best management strategy for each group.

Evaluation of BRCA1 and BRCA2 mutations and risk-prediction models in a typical Asian country (Malaysia) with a relatively low incidence of breast cancer.

INTRODUCTION: The cost of genetic testing and the limited knowledge about the BRCA1 and BRCA2 genes in different ethnic groups has limited its availability in medium- and low-resource countries, including Malaysia. In addition, the applicability of many risk-assessment tools, such as the Manchester Scoring System and BOADICEA (Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm) which were developed based on mutation rates observed primarily in Caucasian populations using data from multiplex families, and in populations where the rate of breast cancer is higher, has not been widely tested in Asia or in Asians living elsewhere. Here, we report the results of genetic testing for mutations in the BRCA1 or BRCA2 genes in a series of families with breast cancer in the multi-ethnic population (Malay, Chinese and Indian) of Malaysia. METHOD: A total of 187 breast cancer patients with either early-onset breast cancer (at age

Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks.

DNA ligases catalyse the joining of DNA single- and double-strand breaks. Saccharomyces cerevisiae Cdc9p is a homologue of mammalian DNA ligase I and is required for DNA replication, recombination and single-strand break repair. The other yeast ligase, Lig4p/Dnl4p, is a homologue of mammalian DNA ligase IV, and functions in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair [1] [2] [3] [4]. Lig4p interacts with Lif1p, the yeast homologue of the human ligase IV-associated protein, XRCC4 [5]. This interaction takes place through the carboxy-terminal domain of Lig4p and is required for Lig4p stability. We show that the carboxy-terminal interaction region of Lig4p is necessary for NHEJ but, when fused to Cdc9p, is insufficient to confer NHEJ function to Cdc9p. Also, Lif1p stimulates the in vitro catalytic activity of Lig4p in adenylation and DNA ligation. Nevertheless, Lig4p is inactive in NHEJ in the absence of Lif1p in vivo, even when Lig4p is stably expressed. We show that Lif1p binds DNA in vitro and, through in vivo cross-linking and chromatin immuno precipitation assays, demonstrate that it targets Lig4p to chromosomal DNA double-strand breaks. Furthermore, this targeting requires another key NHEJ protein, Ku.

Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] [2], XRCC4 and DNA ligase IV [3] [4] [5] [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9] [10] [11]. We have identified a mutation within a highly conserved motif encompassing the active site in DNA ligase IV from 180BR cells. The mutated protein is severely compromised in its ability to form a stable enzyme-adenylate complex, although residual activity can be detected at high ATP concentrations. Our results characterize the first patient with a defect in an NHEJ component and suggest that a significant defect in NHEJ that leads to pronounced radiosensitivity is compatible with normal human viability and does not cause any major immune dysfunction. The defect, however, may confer a predisposition to leukaemia.

p53 Status does not affect photodynamic cell killing induced by hypericin.

PURPOSE: Given that p53 is a tumor suppressor that plays a central role in the cellular response to DNA damage and that more than 50% of all cancers have mutated p53, the wider utility of photodynamic therapy (PDT) in the treatment of cancer will depend on an understanding of whether p53 status modulates response to PDT. In this study, we investigated the photosensitivity of isogenic cell lines that differ only in their p53 status to PDT using hypericin as the photosensitizer. METHODS: Acute (MTT) and chronic (clonogenic) cytotoxic assays were performed on two osteosarcoma cell-lines (U2OS and U2OS+p53DD) that are isogenic except that the latter expresses dominant negative p53. The inducible expression of p53 was determined on western blots. Uptake of hypericin, cell cycle profile analysis, measurement of membrane phosphatidylserine externalization and changes in mitochondrial membrane potential were investigated using flow cytometry. RESULTS: Hypericin uptake was observed to be equivalent in U2OS and U2OS+p53DD cells. There were no significant differences in cell killing between these cell-lines in both the MTT and clonogenic assays (IC(50) of 0.4 microg/ml from MTT assay). p53 expression did not increase up to 24 h after PDT treatment in both cell lines. There were also no significant differences in the cell-cycle arrest profiles and timing of onset of apoptosis. CONCLUSIONS: Taken together, these results suggest that the status of p53 may not be important in PDT-mediated cell killing or induction of apoptosis. By extension, these results imply that PDT may be used with equal efficacy for the treatment of p53-positive and -negative tumors.

First prenatal diagnosis of the carnitine transporter defect.

We report the first attempt at prenatal diagnosis of the carnitine transporter defect in a fetus at high risk of having the disorder. Analysis of cultured CVS after prolonged culture predicted that the fetus was not affected but might be heterozygous for the carnitine transporter defect, but chromosome 15 satellite DNA markers showed no paternal contribution, suggesting that the CVS cells assayed were of predominantly maternal origin. Subsequent assay of cultured amniocytes predicted that the fetus would be affected, and this was confirmed in the newborn period. We conclude that prenatal diagnosis of the carnitine transporter defect is possible, but where results depend on extended culture of CVS, molecular studies should be performed to confirm genetic contributions from both parents.

Person-to-person transmission of cholera in a psychiatric hospital.

An outbreak of cholera caused by Vibrio cholerae O1, biotype el tor, serotype Ogawa, phage type 4, was reported in a psychiatric hospital in Singapore. A total of 74 inmates (18 symptomatic and 56 asymptomatic) were infected; two of them died. Extensive epidemiological investigations showed that the organism was not transmitted by contaminated food or water but through close person-to-person contact. Early recognition of the outbreak and prompt implementation of epidemic control measures comprising surveillance of diarrhoea, rectal swabbing of all asymptomatic inmates, isolation of those found to be infected, maintenance of a high standard of environmental sanitation and mass chemoprophylaxis with doxycycline, rapidly brought the outbreak under control.

Ecthyma gangrenosum in a patient with hypogammaglobulinemia.

Ecthyma gangrenosum is a characteristic skin lesion that is caused by Pseudomonas aeruginosa (P. aeruginosa) in the majority of cases. Systemic P. aeruginosa usually complicates debilitating conditions like leukaemia, burns and cystic fibrosis. We report a patient with underlying hypogammaglobulinemia who developed ecthyma gangrenosum secondary to P. aeruginosa septicaemia, which was potentially life-threatening. Recognition of the characteristic skin lesions with prompt initiation of appropriate antibiotics and intravenous immunoglobulins were life-saving. A review of the English literature reports three other cases of ecthyma gangrenosum in patients with underlying hypogammaglobulinemia.

Tissue-limited mosaicism in Pallister-Killian syndrome -- a case in point.

We report a case of Pallister-Killian syndrome in a term female infant. Antenatal ultrasound showed left diaphragmatic hernia and polyhydramnios. She was ventilated from birth and the diaphragm defect repaired on day 5. She had dysmorphic features, including median cleft palate, patchy frontotemporal alopecia, hypopigmented skin whorls, and bilateral profound sensorineural hearing loss. Fetal and postnatal karyotypes of peripheral lymphocytes were both normal, 46, XX. Subsequently, a skin fibroblast culture showed mosaic tetrasomy of isochromosome 12p both on G-banding and fluorescence in situ hybridization, consistent with Pallister-Killian syndrome. This case illustrates the importance of using the appropriate sample type for karyotype analysis with implications for prenatal and postnatal diagnosis.

Molecular analysis of Hb Q-H disease and Hb Q-Hb E in a Singaporean family.

Hb Q (alpha 74Asp-His) results from a mutation in the alpha-gene such that abnormal alpha Q-chains are synthesized. The alpha Q-chains combine with the normal Beta A-chains to form abnormal Hb alpha 2Q beta 2A (Hb Q). Hb Q-H disease is rare, and has been reported only in the Chinese. We report here a Chinese family, were the mother diagnosed with Hb Q-H disease and the father with Hb E heterozygosity and a child with Hb Q-E-thalassemia. Thalassemia screening of the mother's blood revealed a Hb level of 6.8g/dl with low MCV and MCH. Her blood film was indicative of thalassemia. Cellulose acetate electrophoresis showed Hb H and Hb Q with the absence of Hb A. Globin chain biosynthesis was carried out and alpha Q- and beta-chains were detected. Normal alpha- chains were absent. Digestion of the mother's DNA with Bam HI and Bgl II followed by hybridization with the 1.5 kb alpha-Pst probe showed a two alpha-gene deletion on one chromosome and the -alpha Q chain mutant with the -alpha 4.2 defect on the other chromosome. DNA amplification studies indicated the two-gene deletion to be of the -SEA/ defect. The patient was concluded to possess Hb Q-H disease (--SEA/-alpha 4.2Q). Cellulose acetate electrophoresis of the father's blood showed the presence of Hb A, F and E. Molecular analysis of the father's DNA confirmed an intact set of alpha-genes (alpha alpha/alpha alpha). Globin chain biosynthesis of fetal blood of their child showed gamma, beta A, beta E, alpha A and alpha Q-chains. Molecular analysis of the child's DNA showed one alpha-gene deletion, thus giving a genotype of alpha alpha/-alpha 4.2Q beta beta E.

Cytogenetic abnormalities in childhood acute lymphoblastic leukaemia.

A retrospective study of 50 children newly diagnosed with acute lymphoblastic leukaemia (ALL) from December 1988 to November 1993 was carried out, examining the disease conditions and cytogenetic abnormalities. Of the 50 children, 8 had no cytogenetic examination of the marrow at the time of diagnosis and 7 had a poor yield from the marrow. Fifty percent of the remaining children had a normal karyotype. The rest of the children had hyperdiploidy and structural chromosomal abnormalities (mainly translocation and deletions) in equal proportions. Overall mortality of the whole group was about 26% with 2 lost to follow-up. There were 2 patients who relapsed while on treatment of whom 1 died. Cytogenetic abnormalities were correlated with clinical variables known to have prognostic significance. The group with hyperdiploidy had a significantly lower mean total white count at presentation and none of them died. The group with translocation abnormalities had a lower mean platelet count at presentation. Almost all in the group with hyperdiploidy and a great majority of the other 2 groups with normal or structural cytogenetic abnormalities were of B cell lineage. The median survival times for the hyperdiploid, normal karyotype and translocation patients were 1800 days, 1450 days and 700 days respectively.

Pericentric inversion 9--incidence and clinical significance.

Pericentric inversion of the chromosome 9, inv(9)(p11q13), is such a common occurrence that some cytogeneticists would consider them as normal variants. The incidence is said to be about 1% to 1.65% in the general population. Despite being categorised as a minor chromosomal rearrangement which does not correlate with abnormal phenotypes, many reports in the literature raised conflicting views regarding the association with subfertility and recurrent abortions, abnormal clinical conditions, as well as chromosomal abnormalities arising as a result of having this inversion. We studied the incidence and clinical significance of inv(9)(p11q13) patients retrospectively from 2448 antenatal cytogenetic analysis and 1058 peripheral blood karyotype collected over a 3-year period. Thirty cases of inv(9)(p11q13) from 29 families were found from the antenatal group, which gave an incidence of 1.2% and 6 cases from the peripheral blood karyotype analysis (incidence of 0.6%). The parental origin of the inv(9)(p11q13) in the antenatal group was of equal proportion and there was also no sex predilection for the fetuses carrying the inversion. The babies with the inversion were born with no phenotypic abnormalities. The 6 cases picked up from the peripheral blood karyotype analysis were 2 cases of paediatric patients with associated chromosomal abnormalities (one Trisomy 21 and the other del 13(q22q32)) and 4 adult patients with obstetric and fertility problems. The incidence of subfertility (36%) appeared to be high amongst the adult patients with inv(9)(p11q13). This may represent a true reflection of subfertility in inv(9)(p11q13) patients or a selective bias towards the older subfertile women who conceive at a later age and thus utilising the antenatal diagnostic setup.