Yew Koon Lim

AFP(+), ESC-derived cells engraft and differentiate into hepatocytes in vivo

A major problem in gene therapy and tissue replacement is accessibility of tissue‐specific stem cells. One solution is to isolate tissue‐specific stem cells from differentiating embryonic stem (ES) cells. Here, we show that liver progenitor cells can be purified from differentiated ES cells using alpha‐fetoprotein (AFP) as a marker. By knocking the green fluorescent protein (GFP) gene into the AFP locus of ES cells and differentiating the modified ES cells in vitro, a subpopulation of GFP+ and AFP‐expressing cells was generated. When transplanted into partially hepatectomized lacZ‐positive ROSA26 mice, GFP+ cells engrafted and differentiated into lacZ‐negative and albumin‐positive hepatocytes. Differentiation into hepatocytes also occurred after transplantation of GFP+ cells in apolipoprotein‐E‐ (ApoE) or haptoglobin‐deficient mice as demonstrated by the presence of ApoE‐positive hepatocytes and ApoE mRNA in the liver of ApoE‐deficient mice or by haptoglobin in the serum and haptoglobin mRNA in the liver of haptoglobin‐deficient mice. This study describes the first isolation of ES‐cell‐derived liver progenitor cells that are viable mediators of liver‐specific functions in vivo.

Expression of major HDL-associated antioxidant PON-1 is gender dependent and regulated during inflammation.

Paraoxonase 1, an HDL-associated enzyme that confers antioxidant activity on HDL, and its activity in serum have been correlated with protection against atherosclerosis, an oxidative disease. However, serum PON-1 activity is highly variable and its regulation is complex, involving both genetic and environmental factors. It is influenced by gender and inflammation, two important factors in atherosclerosis. Serum PON-1 activity has been shown to be lower in male mice and is decreased in male Syrian hamster during inflammation. Here we show that male mice had lower hepatic PON-1 mRNA that increased by 170% after castration. Our data also suggested that this effect was testes but not plasma testosterone dependent. Ovariectomy had no effect on PON-1 mRNA in female mice. LPS caused hepatic PON-1 mRNA to decrease further in male mice, and to increase moderately in female mice. Anti-inflammatory dexamethasone enhanced PON-1 mRNA level by 2-fold in male and female LPS-treated mice, and increased PON-1 expression by 8-fold in Hepa cell, a mouse hepatoma cell line. Therefore, antioxidant PON-1 is regulated at the mRNA level in a gender-specific manner by proinflammatory LPS and anti-inflammatory dexamethasone.

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.

Gonadal effects on plasma ACE activity in mice

Angiotensin-converting enzyme (ACE) regulates blood pressure and is an important target in the management of hypertension. Hypertension is a gender biased disease. Plasma ACE activity is significantly higher in male mice (309 U/l) than female mice (237 U/l) and is reduced significantly upon gonadectomy to 224 and 209 U/l, respectively. Although, the gonads influence plasma ACE activity in both male and female mice, the effect is more pronounced in male mice. Plasma ACE is derived from the cleavage of tissue ACE and lung has the highest concentration of tissue ACE. However, lung ACE activity is not gender dimorphic but increases significantly upon gonadectomy in both male and female. ACE mRNA level in the lung is not influenced by gender or gondaectomy. Therefore, the gonads affect plasma ACE activity by influencing cleavage of tissue ACE to plasma ACE and/or decrease stability of plasma ACE in gonadectomized mice is mediated.

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.

The Role of Vitamin D Receptor Polymorphisms in Predicting the Response to Therapy for Nonmuscle Invasive Bladder Carcinoma

Purpose: Clinical and pathological predictors of bladder carcinoma recurrence and progression are relatively well defined. However, there is a paucity of genetic data specifically on the association of single nucleotide polymorphisms in specific genes for predicting recurrence and progression following immunotherapy. The VDR gene was found to regulate the immunomodulatory effects of vitamin D and it enhances the innate immunity system. We evaluated 3 VDR single nucleotide polymorphisms and their predictive role on the response to immunotherapy. Materials and Methods: Patients with bladder cancer at intermediate‐high risk who underwent post‐transurethral resection intravesical bacillus Calmette‐Guérin in Singapore and Hong Kong from 1995 to 2014 were recruited for analysis. We evaluated 3 VDR single nucleotide polymorphisms using polymerase chain reaction. Kaplan‐Meier survival curves and relationships with outcomes were analyzed by multivariable Cox regression. Results: A total of 338 predominantly Chinese patients were included in study. Individuals carrying the VDR genotype Bsm A/G were significantly associated with lower time to recurrence after bacillus Calmette‐Guérin therapy (p <0.001). On multivariable analysis the HR of recurrence in patients with the Bsm A allele was 3.95 times that in patients without the allele (p = 0.037). Patients with the VDR GATC subhaplotype were 3.05 times more likely than patients with other subhaplotypes to experience recurrences (p = 0.003). Study limitations include the small sample size and the lack of information on previous bacillus Calmette‐Guérin vaccine exposure and on vitamin D levels. Conclusions: Our findings in this study suggest that various VDR single nucleotide polymorphisms are associated with recurrences after bacillus Calmette‐Guérin immunotherapy. Further functional studies should be performed to elucidate the significance of the VDR gene in the management of bladder cancer and the potential therapy implications.

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.

MP15-17 BEYOND SUPPLEMENTATION: THE ROLE OF VITAMIN D IN NON-MUSCLE INVASIVE BLADDER CANCER

patients (OR 1.77; 95%CI 1.09-2.85; p1⁄40.02). Estimated 5-yr RFS, CSS, and OS in patients <70 versus 70 was 70% vs 62% (p1⁄40.14), 84% vs 77% (p1⁄40.06), and 74% vs 54% (p<0.01). On multivariable Cox regression analyses, age 70 was not independently associated with RFS (HR 1.10; 95% CI 0.79-1.52; p1⁄40.57) or CSS (HR 1.22; 95% CI 0.82-1.82; p1⁄40.32), but remained associated with decreased OS (HR 1.91; 95% CI 1.50-2.45; p<0.01). Moreover, among all patients and as stratified by age, pathologic upstaging was associated with worse RFS, CSS, and OS on both multivariable Cox proportional hazards regression and in competing risk (of non-cancer death) models (Table). CONCLUSIONS: Older patients with NMIBC had similar risks of pathologic upstaging at RC, and patients upstaged at surgery had inferior cancer outcomes across age strata. Meanwhile, advanced age was not associated with increased risks of PBT, pORT, or perioperative complications. These data support the use of RC for select older patients with high-risk NMIBC.