Jen-Hwei Sng

The prevalence of BRCA1 mutations in Chinese patients with early onset breast cancer and affected relatives

The purpose of this study was to determine the prevalence of BRCA1 mutations in Chinese breast cancer patients in Singapore. BRCA1 analysis was conducted in consecutive patients with breast cancer before the age of 40 years (76 women), or whose relatives had breast or ovarian cancer (16 women). Ten patients had both early onset breast cancer and affected relatives. Genomic DNA from peripheral mononuclear blood cells was studied by using the protein transcription–translation assay (exon 11) and single-strand conformational polymorphism, with subsequent DNA sequencing. All six disease-causing mutations occurred in women under 40 years (8.6%) with three occurring in patients under 35 years (three out of 22 patients, 13.6%). Mis-sense mutations of unknown significance were found in three patients. Two of the ten women with affected relatives under 40 years had BRCA1 mutations. The prevalence of BRCA1 mutations in Chinese patients with early onset breast cancer is similar to that observed in Caucasian women. Most Chinese patients with affected relatives were not carriers of BRCA1 mutations.

Identification of novel BRCA large genomic rearrangements in Singapore Asian breast and ovarian patients with cancer.

Large genomic rearrangements have been reported to account for about 10–15% of BRCA1 gene mutations. Approximately, 90 BRCA rearrangements have been described to date, all of which but one have been reported in Caucasian populations of predominantly Western European descent. Knowledge of BRCA genomic rearrangements in Asian populations is still largely unknown. In this study, we have investigated for the presence of BRCA rearrangements among Asian patients with early onset or familial history of breast or ovarian cancer. Using multiplex ligation‐dependent probe amplification (MLPA), we have analyzed 100 Singapore patients who previously tested negative for deleterious BRCA mutations by the conventional polymerase chain reaction‐based mutation detection methods. Three novel BRCA rearrangements were detected, two of which were characterized. The patients with the rearrangements, a BRCA1 exon 13 duplication, a BRCA1 exon 13–15 deletion and a BRCA2 exon 4–11 duplication, comprise 3% of those previously tested negative for BRCA mutations. Of the BRCA1 and BRCA2 pathogenic mutations identified in our studies on Asian high‐risk breast and ovarian patients with cancer to date, these rearrangements constitute 2/19 and 1/2 of the BRCA1 and BRCA2 pathogenic mutations, respectively. Given the increasing number of rearrangements reported in recent years and their contribution to the BRCA mutation spectrum, the presence of BRCA large exon rearrangements in Asian populations should be investigated where clinical, diagnostic service is recommended.

Prevalence of PALB2 Mutations in Breast Cancer Patients in Multi-Ethnic Asian Population in Malaysia and Singapore

Background The partner and localizer of breast cancer 2 (PALB2) is responsible for facilitating BRCA2-mediated DNA repair by serving as a bridging molecule, acting as the physical and functional link between the breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) proteins. Truncating mutations in the PALB2 gene are rare but are thought to be associated with increased risks of developing breast cancer in various populations. Methods We evaluated the contribution of PALB2 germline mutations in 122 Asian women with breast cancer, all of whom had significant family history of breast and other cancers. Further screening for nine PALB2 mutations was conducted in 874 Malaysian and 532 Singaporean breast cancer patients, and in 1342 unaffected Malaysian and 541 unaffected Singaporean women. Results By analyzing the entire coding region of PALB2, we found two novel truncating mutations and ten missense mutations in families tested negative for BRCA1/2-mutations. One additional novel truncating PALB2 mutation was identified in one patient through genotyping analysis. Our results indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the Malaysian and Singaporean populations.

Comprehensive spectrum of BRCA1 and BRCA2 deleterious mutations in breast cancer in Asian countries

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.

NRAMP1 and hGPX1 Gene Polymorphism and Response to Bacillus Calmette-Guérin Therapy for Bladder Cancer

BACKGROUND The natural resistance-associated macrophage protein 1 (NRAMP1) gene is associated with susceptibility to Mycobacterium tuberculosis in humans and to bacillus Calmette-Guérin (BCG) in mice. The detoxification enzyme, human glutathione peroxidase 1 (hGPX1), is associated with recurrence of bladder cancer (BCa). OBJECTIVE To determine whether NRAMP1 and hGPX1 gene polymorphisms correlate with response to BCG immunotherapy for non-muscle-invasive BCa (NMIBC). DESIGN, SETTING, AND PARTICIPANTS DNA was obtained from the peripheral blood of 99 NMIBC patients who were prospectively randomized to receive postresection intravesical BCG (81 mg [n=50] or 27 mg [n=19]) or BCG (27 mg) with interferon alpha (IFN-α; n=30). The median follow-up time was 60 mo. INTERVENTION Intravesical BCG or BCG-IFN-α. MEASUREMENTS Restriction fragment length polymorphism (RFLP) analysis was performed to identify polymorphisms in the NRAMP1 promoter region (GT repeat number) and at position 543 (aspartate [D] and/or asparagine [N] expression) within the NRAMP1 protein (D543N) and position 198 (proline and/or leucine expression) within the hGPX1 protein (Pro198Leu). Data were analyzed using χ(2) analysis, multivariate analysis, and Kaplan-Meier curves. RESULTS AND LIMITATIONS On univariate analysis, the NRAMP1 D543N G:G genotype had decreased cancer-specific survival (CSS; p=0.036). The hGPX1 CT genotype (Pro-Leu) had decreased recurrence time (p=0.03) after BCG therapy. On multivariate analysis, patients with the NRAMP1 D543N G:G genotype and allele 3 (GT)n polymorphism had decreased recurrence time (p=0.014 and p=0.03) after BCG therapy. The limitation of this study was its small sample size. CONCLUSIONS Polymorphisms of the NRAMP1 and hGPX1 genes may be associated with recurrence of BCa after BCG immunotherapy.

Are medullary breast cancers an indication for BRCA1 mutation screening? A mutation analysis of 42 cases of medullary breast cancer.

Recommended guidelines have limited breast cancer gene (BRCA1) mutation testing to individuals with a personal or family history of early onset breast and/or ovarian cancer, and those with multiple affected close relatives. Such large breast cancer families are rare in the general population, limiting the clinical application of the BRCA1 discovery. Previous reports have suggested an association between medullary breast cancer and BRCA1 mutation carriers. To test the feasibility of using these rare histological subtypes as an alternative to epidemiological factors, 42 cases of medullary cancer unselected for family history were screened for BRCA1 point mutations and large exon rearrangements. The large majority (83%) of these patients did not have significant family of breast or ovarian cancer. Two deleterious mutations resulting in a premature stop codon, and one exon 13 duplication were found. All mutations were detected in patients with typical medullary cancer, who had family history of multiple breast and ovarian cancers. Our findings suggest that medullary breast cancers are not an indication for BRCA1 mutation screening in the absence of significant family risk factors.

Cancer-specific methylation in the BRCA1 promoter in sporadic breast tumours

To the EditorSilencing of the BRCA1 gene via promoter hyperme-thylation has been proposed as one mechanism for itsinactivation and found to occur in 10–31% of all sporadicbreast tumours [1, 2]. The impact of promoter hyperme-thylation on the BRCA1 gene is similar to that of a germ-line BRCA1 mutation in terms of gene expression patterns[3, 4]. Breast cancer tumours, with extensive hyperme-thylation in the BRCA1 promoter, have consistently beencorrelated with reduced BRCA1 expression [2, 5].Previous studies have shown a region of 267 bp (-224to ?43; GenBank for BRCA1, U37574) within the BRCA1promoter to be functionally important for its optimaltranscriptional regulation. In particular, deletion of a 222bp (-220 to ?20) fragment within this region, termed thepositive regulatory region (PRR), has been shown to resultin complete loss of the BRCA1 promoter activity [6, 7].Alterations by DNA methylation are thought to be animportant event in tumour initiation; hence, the identifi-cation of tumour-specific methylation patterns in BRCA1promoter may serve as targets for breast cancer detection.The aim of this study was to investigate the presence ofcancer-specific methylation within the PRR of BRCA1 insporadic breast tumours. Histologically proven archivalmatched breast cancer tissues, as well as normal breasttissues from the affected breast, from 23 sporadic breastcancer cases were selected, and their use had the approvalof the institutional ethical committee, National UniversitySingapore-Institutional Review Board (NUS-IRB). Mat-ched tumour and corresponding normal tissues of the samepatient were used to compensate for any age-relatedmethylation [8, 9].BRCA1 promoter hypermethylation was detected in34.8% (8/23) of Asian sporadic breast tumours using MS-PCR-based method (Fig. 1a, b). MS-PCR was carried outat least twice for all samples to confirm the percentage ofcases with BRCA1 promoter hypermethylation. Differencein BRCA1 methylation levels between each matchedtumour and normal tissue was initially determined by cal-culating the mean, standard deviation and 95% CI of the 23matched samples. Tumour samples with difference inBRCA1 methylation levels greater than the upper value of95% CI were considered to be hypermethylated. In addi-tion, methylation in the BRCA1 promoter was alsoobserved in normal breast tissues from breast cancerpatients, an observation similarly reported in a previousstudy [10].Our data also showed that breast tumours with BRCA1hypermethylation exhibited a significant loss of BRCA1transcripts (Fig. 1c). BRCA1 hypermethylated tumourswere then screened for BRCA1 mutation where the entirecoding sequence, including splice junctions and neigh-bouring intronic regions of BRCA1 were analysed usingpreviously described methods [11]. This was carried out toexclude somatic mutation as the cause for loss of BRCA1expression in these samples. No mutations were found thussuggesting BRCA1 transcriptional silencing by promoterhypermethylation in these tumours. MS-PCR products

BRCA1 disease-associated haplotypes in Singapore Malay women with early-onset breast/ovarian cancer.

Keywords BRCA1 Haplotype Early-onset Breast OvarianTo the EditorFounder mutations are recurrent mutations with sharedcommon haplotypes arising either from a population that isestablished by a small group of individuals or from a sit-uation where the population size has decreased drastically[1]. Common shared haplotypes have been determined byanalyzing microsatellite markers in various populationgenetics studies [2].BRCA1 c.66–67delAG mutation is the most frequentlyreported mutation in about 1% of the Ashkenazi Jews andreconstruction of haplotypes in carriers has demonstratedstrong evidence for founder effects [3–6]. Identification offounder mutations has important clinical applications as itallows for a more accurate assessment on prevalence andpenetrance of BRCA1 mutations in the clinical settings [1].The Singapore Malays are a race of people of Malay orIndonesian origins and constitute 14% of Singapore’s 2.7million population [7]. Singapore Malays share genetic,cultural and historical links with Malays in PeninsularMalaysia, the coast of Borneo, on the east coast ofSumatra and the smaller islands that lie between theseareas and extends to the Southern Philippines with anestimated population of 18 million or more [8]. Virtuallyall Singapore Malays are Muslim, and their religionreflects the socio-cultural practices of this group [9]. TheBRCA1 deleterious mutation, c.2845insA, was firstreported in 2/7 unrelated Singapore Malay patients indi-cating the possible presence of a founder mutation [10].Two follow-on studies on the Singapore Malays havesince led to the identification of a common sharedhaplotype at BRCA1 intragenic markers in all BRCA1c.2845insA carriers, strongly suggesting a commonancestral origin for this mutation [11–12]. In these stud-ies, we identified the BRCA1c.2845insA mutation in 6/62(9.7%) unrelated early-onset breast/ovarian cases and 1fallopian tube cancer patient. Haplotype analysis per-formed at 3 BRCA1 intragenic polymorphic markers(D17S855, D17S1322 and D17S1323) indicated a sharedhaplotype, 142-127-159, in all BRCA1 c.2845insA muta-tion carriers [11, 13].In this study, 78 early-onset breast and ovarian cancerpatients, diagnosed before the ages of 45 and 50 yearsrespectively were recruited from the National UniversityHospital, Singapore, together with 164 unrelated age-matched normal healthy controls. This study had theinstitutional ethical committee approval and signed writ-ten informed consent was obtained from each participantbefore peripheral blood collection. All cancerous speci-mens were screened for the presence of the founder

MP65-05 IFNα MODULATES THE RESPONSE TO BCG IMMUNOTHERAPY IN BLADDER CANCER PATIENTS WITH SPECIFIC CTLA4 SINGLE NUCLEOTIDE POLYMORPHISMS

respect to the GSTT2B genotypes. The impact of BCG instillation (numbers) on recurrence was analyzed in a subset of patients for whom complete 10y follow-up data was available. Analysis was performed using SPSS 23.0 and p<0.05 was taken to be significant. RESULTS: GSTT2 was silenced in MGH cells (GSTT2B homozygous full length (GSTT2B)) and overexpressed in UMUC3 and U937 cells (GSTT2B homozygous deleted (GSTT2B)). A 2h exposure to BCG resulted in decreased ROS in GSTT2 silenced cells (p<0.05) and increased ROS in GSTT2 overexpressing cells (p<0.05). There was no difference in cellular cytotoxicity to BCG with respect to GSTT2 expression. However, intracellular BCG survival increased at 2 hours when GSTT2 was silenced (p<0.05) and decreased when GSTT2 was overexpressed (p<0.05). There was no significant difference between these groups at 24h. The majority of patients with complete 10y follow-up data, completed a 6+3 BCG schedule (n1⁄463) and n1⁄422 had less than 8 instillations. Patients with GSTT2B genotype (n1⁄46) who received 8 or less BCG instillations, were recurrence free (Likelihood ratio 1⁄4 0.040, p1⁄40.054). In the group that received at least 9 instillations of BCG, the GSTT2B was associated with earlier recurrence. CONCLUSIONS: GSTT2 expression decreases cellular ROS and BCG survival. GSTT2B was associated with lower likelihood of recurrence for patients who received 8 or less BCG instillations. In contrast patients with GSTT2B who received 9 or more instillations of BCG had earlier recurrences. Hence GSTT2B could be used as a marker for patients who will do well with less BCG therapy.