Genome-wide association studies for pelvic organ prolapse in the Japanese population.

Pelvic organ prolapse (POP) affects approximately 40% of elderly women, characterized by the descent of the pelvic organs into the vaginal cavity. Here we present the results of a genome-wide association study (GWAS) for susceptibility to POP comprising 771 cases and 76,625 controls in the Japanese population. We identified a significant association of WT1 locus with POP in the Japanese population; rs10742277; odds ratio (OR) = 1.48, 95% confidence interval (CI), 1.29-1.68, P = 6.72 × 10-9. Subsequent cross-ancestry GWAS meta-analysis combining the Japanese data and previously reported European data, including 28,857 cases and 622,916 controls, identified FGFR2 locus as a novel susceptibility locus to POP (rs7072877; OR = 1.06, 95% CI, 1.04-1.08, P = 4.11 × 10-8). We also observed consistent directions of the effects for 21 out of 24 European GWAS derived loci (binomial test P = 2.8 × 10-4), indicating that most of susceptibility loci for POP are shared across the Japanese and European populations.

Increased level of serum leucine-rich-alpha-2-glycoprotein 1 in patients with clear cell renal cell carcinoma.

BACKGROUND: Currently, no useful serum markers exist for clear cell renal cell carcinoma (ccRCC), making early detection challenging as diagnosis relies solely on imaging tests. Radiation exposure is also a concern due to multiple required CT examinations during treatment. Renal cell carcinoma (RCC) histological types include ccRCC and non-clear cell RCC (non-ccRCC); however, treatment response to medications varies which necessitates accurate differentiation between the two. Therefore, we aimed to identify a novel serum marker of RCC. Increased LRG1 expression in the serum has been demonstrated in multiple cancer types. However, the expression of LRG1 expression in the serum and cancer tissues of patients with RCC has not been reported. Since ccRCC is a hypervascular tumor and LRG1 is capable of accelerating angiogenesis, we hypothesized that the LRG1 levels may be related to ccRCC. Therefore, we examined LRG1 expression in sera from patients with RCC. METHODS: Using an enzyme-linked immunosorbent assay, serum levels of leucine-rich-alpha-2-glycoprotein 1 (LRG1) were measured in 64 patients with ccRCC and 22 patients non-ccRCC who underwent radical or partial nephrectomy, as well as in 63 patients without cancer. RESULTS: Median values of serum LRG1 and their inter-quartile ranges were 63.2 (42.8-94.2) µg/mL in ccRCC, 23.4 (17.7-29.6) µg/mL in non-ccRCC, and 36.0 (23.7-56.7) µg/mL in patients without cancer, respectively (ccRCC vs. non-ccRCC or patients without cancer: P < 0.001). C-reactive protein (CRP) levels (P = 0.002), anemia (P = 0.037), hypercalcemia (P = 0.023), and grade (P = 0.031) were independent predictors of serum LRG1 levels in ccRCC. To assess diagnostic performance, the area under the receiver operating characteristic curve of serum LRG1 was utilized to differentiate ccRCC from non-cancer and non-ccRCC, with values of 0.73 (95% CI, 0.64-0.82) and 0.91 (95% CI, 0.82-0.96), respectively. CONCLUSIONS: LRG1 served as a serum marker associated with inflammation, indicated by CRP, anemia, hypercalcemia, and malignant potential in ccRCC. Clinically, serum LRG1 levels may assist in differentiating ccRCC from non-ccRCC with excellent diagnostic accuracy.

Stage-specific Embryogenic Antigen-4 Expression in Castration-resistant Prostate Cancer and its Correlation With the Androgen Receptor.

BACKGROUND/AIM: Stage-specific embryonic antigen (SSEA)-4 plays important roles in the malignant aggressiveness of various cancers. The aim of this study was to investigate the pathological characteristics of SSEA-4 in castration-resistant prostate cancer (CRPC). MATERIALS AND METHODS: SSEA-4 expression and its pathological roles were evaluated in five prostate cancer (PC) cell lines and 27 CRPC tissues. The relationship between SSEA-4 and the androgen receptor (AR) was also examined. RESULTS: SSEA-4 expression was detected in AR-negative cells (PC3, DU145, and AICaP1) but was not detected in AR-positive cells (LNCaP and AICaP2). SSEA-4 expression in human CRPC tissues was significantly higher than that in locally advanced or metastatic hormone sensitive PC (HSPC) tissues. A negative correlation was also detected between SSEA-4 and AR in CRPC tissues but not in HSPC tissues. CONCLUSION: SSEA-4 was over-expressed in CRPC and the changes were mediated by complex mechanisms that related to the AR and hormonal therapy.

Collagen type 1A1, type 3A1, and LOXL1/4 polymorphisms as risk factors of pelvic organ prolapse.

OBJECTIVE: Collagen and elastin are the main components of the female pelvic tissue. We investigated whether single nucleotide polymorphisms (SNPs) of collagen type 1 alpha 1 (COL1A1), collagen type 3 alpha 1 (COL3A1), and lysyl oxidase-like (LOXL) 1 and 4 were associated with the onset of pelvic organ prolapse (POP) in Japanese women. Fifty-two women with POP and 28 women without POP were included. SNPs were identified using the TaqMan® SNP genotyping assay. RESULTS: Age, parity, and lower urinary tract symptoms were significantly higher in the POP group than in the control group. The prevalence of genotypes with rs2862296 polymorphism of LOXL4, an enzyme essential for extracellular matrix remodeling, was different between the POP (26.9% for GG, 51.9% for AG) and control groups (14.8% for GG, 33.3% for AG). However, polymorphisms of COL1A1, COL3A1, and LOXL1 were not related to the onset of POP. In the multivariate logistic regression analysis, age was significantly associated with the occurrence of POP. In the univariate analysis, LOXL4 polymorphism was associated with the onset of POP in Japanese women. The knowledge of acquired risk factors and polymorphisms in the genomic background of patients with POP may help prevent POP via early conservative interventions.

Urine Lactoferrin as a Potential Biomarker Reflecting the Degree of Malignancy in Urothelial Carcinoma of the Bladder.

Urothelial carcinoma of the bladder (UCB) is potentially life-threatening; therefore, we aimed to discover a novel urine biomarker for diagnosis and prognostication of UCB. This is a retrospective case-control study. Exploration of a new biomarker using urine from 20 UCB patients in the present study revealed that urinary level of lactoferrin (LF), a multifunctional glycoprotein released from neutrophils, was higher in 11 of 15 with invasive/high-grade UCB than 5 with non-invasive one, and 2 healthy adults. We therefore focused on LF and assessed the value of urine LF normalized by urine creatinine concentration (LF/Cr) using an enzyme-linked immunosorbent assay. Diagnostic performance of urine LF/Cr was examined using urine from 92 patients with primary (newly diagnosed) untreated UCB and 166 controls without UCB, including 62 patients with pyuria, and 104 subjects without pyuria consisting of 84 patients and 20 healthy adults. However, the diagnostic accuracies were accompanied by the risk of bias. In 92 primary UCB patients, both pyuria and tumor-infiltrating neutrophils (TINs) were independent predictors for urine LF/Cr. In contrast, TINs or urine LF/Cr were independent predictors for invasive histology, whereas pyuria was not. In terms of prognostication, urine LF/Cr and nodal metastasis were independent predictors of disease-specific survival in 22 patients with muscle-invasive bladder cancer, characterized by a high mortality rate, in the Cox proportional hazards model. In conclusion, urine LF/Cr linked to TINs was a predictor of both invasive histology and prognosis in UCB. Urine LF/Cr is a potential biomarker reflecting the degree of malignancy in UCB.

Relationship Between Stage-specific Embryonic Antigen-4 and Anti-cancer Effects of Neoadjuvant Hormonal Therapy in Prostate Cancer.

BACKGROUND/AIM: Stage-specific embryonic antigen-4 (SSEA-4) expression is associated with malignant aggressiveness and is useful as a marker for identifying cancer stem cells. Our aim was to assess the relationship between hormonal therapy and SSEA-4 expression in prostate cancer (PC). MATERIALS AND METHODS: SSEA-4 expression in paired specimens from PC patients who underwent neoadjuvant hormonal therapy (NHT) and radical prostatectomy (60 pre-NHT specimens and 60 post-NHT specimens) was evaluated using immunohistochemistry. Proliferation index (PI) and apoptotic index (AI) were also evaluated. RESULTS: Post-NHT tissues had significantly elevated SSEA-4 expression whereas anti-tumor effects of NHT were inversely correlated with SSEA-4 expression level. SSEA-4 expression in post-NHT tissues was significantly associated with biochemical recurrence-free survival. SSEA-4 expression in the post-NHT tissues was positively associated with PI and negatively done with AI. CONCLUSION: SSEA-4 is a potential therapeutic target for limiting the malignant potential in hormone-naïve PC when considering the use of NHT.

Stage-specific embryonic antigen-4 is a histological marker reflecting the malignant behavior of prostate cancer.

Stage-specific embryonic antigen-4 (SSEA-4), a specific marker for pluripotent stem cells, plays an important role in the malignant behavior of several cancers. Here, SSEA-4 expression was evaluated by immunohistochemistry using monoclonal antibody RM1 specific to SSEA-4 in 181 and 117 prostate cancer (PC) specimens obtained by biopsy and radical prostatectomy (RP), respectively. The relationships between SSEA-4 expression in cancer cells or the presence of SSEA-4-positive tumor-infiltrating immune cells (TICs) and clinicopathological parameters were analyzed. SSEA-4 expression in cancer cells was significantly associated with Gleason score, local progression, and lymph node and distant metastasis. In RP specimens, high SSEA-4 expression in cancer cells and the presence of SSEA-4-positive TICs were significant predictors of pT3, i.e., invasion and worse biochemical recurrence (BCR) after RP, respectively, in univariate analysis. In contrast, combination of high SSEA-4 expression in cancer cells and the presence of SSEA-4-positive TICs was an independent predictor for pT3 and BCR in multivariate analysis. Biologically this combination was also independently associated with suppression of apoptosis. Thus, the co-expression of SSEA-4 in cancer cells and TICs may have crucial roles in the malignant aggressiveness and prognosis of PC. Invasive potential and suppression of apoptosis may be linked to SSEA-4 expression.

Prognostic value of the ubiquitin ligase carboxyl terminus of the Hsc70-interacting protein in postmenopausal breast cancer.

The carboxyl terminus of the Hsc70-interacting protein (CHIP) is considered to induce the ubiquitination and degradation of several oncogenic proteins, and play a role in the inhibition of tumor progression and invasion under experimental conditions. However, the impact of CHIP expression on the prognosis of breast cancer patients has not yet been established. In this study, using an immunohistochemical method, 272 patients with invasive breast cancer were assessed for the expression of CHIP (graded scores 0-3) and the statuses of biomarkers, such as estrogen receptor (ER), progesterone receptor (PgR), and HER2. The relationships between the statuses of CHIP and biomarkers as well as clinical features were also evaluated, and that between the expression of CHIP and patient prognosis was analyzed. We revealed that the strong expression of CHIP correlated with positive ER (P < 0.001), positive PgR (P < 0.001), and negative HER2 (P = 0.02). In postmenopausal patients, relapse-free survival (RFS) was significantly better in the high CHIP group than in the low CHIP group (P = 0.042). In addition, RFS and cancer-specific survival (CSS) were significantly better in patients with ER-positive/CHIP score 3 tumors than in those with ER-negative/CHIP score 0 tumors (RFS: P = 0.038, CSS: P = 0.0098). The methylation status of CHIP gene promoter did not always account for the down-regulation of its expression. In conclusion, the overexpression of CHIP is a potent prognostic factor of a good prognosis in ER-positive breast cancer patients in the postmenopausal phase.

ER-activating ability of breast cancer stromal fibroblasts is regulated independently of alteration of TP53 and PTEN tumor suppressor genes.

Carcinoma-associated fibroblasts (CAFs) are associated with tumor progression and metastasis, and are able to activate estrogen receptor (ER) in breast cancer. We established a stable transformant of a human breast cancer cell line to detect CAF-specific ER-activating ability, and found that this CAF ability varied among tumors. Some studies have reported a high frequency of alterations among tumor suppressor genes in stromal cells, but do not generally agree as to the frequency. Moreover, the activation mechanism of CAF-induced estrogen signals, including the effects of these gene aberrations, is not fully understood. We investigated the relevance of tumor suppressor gene aberrations and ER-activating ability in CAFs derived from 20 breast cancer patients. Although CAF-specific ER-activating abilities varied among individual cases, all CAFs maintained wild-type alleles for TP53 and PTEN. Also, copy number aberrations in these genes were not observed in any CAFs. Our results suggest that the ER-activating ability of the CAFs is regulated independently of aberrations in these genes; and that other mechanisms of tumor-stromal interaction may affect activation of estrogen signals in breast cancer.

Differential detection of KRAS mutations in codons 12 and 13 with a modified loop-hybrid (LH) mobility shift assay using an insert-type LH-generator.

BACKGROUND: The loop-hybrid mobility shift assay (LH-MSA) was previously developed for the rapid detection of the EGFR mutation L858R for predicting clinical responses to gefitinib in lung cancer. Recently, clinical importance of determining KRAS mutations has been demonstrated in colorectal tumors as tumors harboring mutated KRAS genes were not responsive to therapy with EGFR-targeted antibodies such as cetuximab. METHODS: We developed a new version of the LH-MSA using an insert-type LH generator that was capable of detecting all 12 KRAS mutations in codons 12 and 13. RESULTS: Feasibility evaluation was performed with this new LH-MSA on 215 colorectal cancer specimens. KRAS codon 12 mutations were detected in 23% specimens and codon 13 mutations in 6.5% specimens by LH-MSA at a rate better than by direct sequencing. CONCLUSIONS: Using the new method, the G13D mutation was readily distinguishable from other KRAS mutations in codon 12 and, therefore, would be advantageous for clinical applications.

Rare MDM4 gene amplification in colorectal cancer: The principle of a mutually exclusive relationship between MDM alteration and TP53 inactivation is not applicable.

MDM4, a homolog of MDM2, is considered a key negative regulator of p53. Gene amplification of MDM4 has been identified in a variety of tumors. MDM2 or MDM4 gene amplification is only associated with the wild-type TP53 gene in retinoblastomas, thus the amplification of the two genes is mutually exclusive. Previously, we demonstrated that MDM2 amplification and TP53 alteration were not mutually exclusive in colorectal cancer, and we identified a subset of colorectal cancer patients without alterations in either the TP53 or the MDM2 gene. In this study, we investigated the gene amplification status of MDM4 in the same set of colorectal cancer cases. Unexpectedly, MDM4 amplification was rare, detected in only 1.4% (3 out of 211) of colorectal cancer cases. All the three gene-amplified tumors also harbored TP53-inactivating mutations. This contradicts the simple mutually exclusive relationship observed in retinoblastomas. Surprisingly, two of the three MDM4-amplified tumors also demonstrated MDM2 amplification. Paradoxically, the MDM4 protein levels were decreased in the tumor tissue of the gene-amplified cases compared with levels in the matched normal mucosa. We speculate that MDM4 might play a role in colorectal carcinogenesis that is not limited to negative regulation of p53 in combination with MDM2. The functional significance of MDM4 is still unclear and further studies are needed.

MDM2 gene amplification in colorectal cancer is associated with disease progression at the primary site, but inversely correlated with distant metastasis.

MDM2 is a crucial negative regulator of the TP53 tumor suppressor and almost 10% of human tumors exhibit MDM2 amplification. Although TP53 pathway perturbation has been extensively examined in colorectal cancer (CRC), only one previous report has evaluated MDM2 amplification in relation to clinicopathological factors. In that report, MDM2 amplification was shown to be associated with disease progression from Dukes' Stages A to D. In this study, we investigated MDM2 amplification by quantitative PCR and fluorescence in situ hybridization (FISH) together with the SNP309 genotypes, and analyzed the correlations with TP53 and KRAS mutations and clinicopathological features in 211 Japanese CRC patients. MDM2 amplification was detected in 8% of the specimens and its incidence was significantly higher in Dukes' stage C than in the combined earlier Stages A and B (P = 0.025). Unexpectedly, the incidence was significantly decreased in Stage D metastatic disease (P = 0.043). The copy number gain ranged from four to eight copies and was generally concordant with gain of centromere 12 using FISH analysis. Together with the results of centromere 1 FISH and TP53 copy number assessment, the MDM2 increment most likely resulted from chromosome 12 gain. The mechanism of the copy number gain and incidence in Dukes' Stage D differed considerably from the previous report. Ethnic or geographic factors could be responsible for these differences. Several promising therapeutic strategies targeting the TP53-MDM2 system are being developed. Further understanding of the significance of MDM2 and MDM2 amplification in CRC is required to facilitate personalized treatment for CRC patients.

Identification of colorectal cancer patients with tumors carrying the TP53 mutation on the codon 72 proline allele that benefited most from 5-fluorouracil (5-FU) based postoperative chemotherapy.

BACKGROUND: Although postoperative chemotherapy is widely accepted as the standard modality for Dukes' stage C or earlier stage colorectal cancer (CRC) patients, biomarkers to predict those who may benefit from the therapy have not been identified. Previous in vitro and clinical investigations reported that CRC patients with wild-type p53 gene (TP53)-tumors benefit from 5-fluorouracil (5-FU) based chemotherapy, while those with mutated TP53-tumors do not. However, these studies evaluated the mutation-status of TP53 by immunohistochemistry with or without single-strand conformation polymorphism, and the mutation frequency was different from study to study. In addition, the polymorphic status at p53 codon 72, which results in arginine or proline residues (R72P) and is thought to influence the function of the protein significantly, was not examined. METHODS: To evaluate the significance of the TP53 mutation as a molecular marker to predict the prognosis of CRC patients, especially those who received postoperative chemotherapy, we examined the mutation by direct sequencing from fresh CRC tumors and evaluated the R72P polymorphism of the mutated TP53 by a combined mutant allele- and polymorphic allele-specific polymerase chain reaction (PCR). RESULTS: The TP53 mutation occurred in 147 (70%) of 211 Japanese CRC tumors. The mutation was observed in 93 (63%) tumors on the R72 allele and in 54 (37%) tumors on the P72 allele. Although the alterations to TP53 have no prognostic significance for CRC patients overall, we found that Dukes' stage C CRC patients who did not receive postoperative chemotherapy and carried the mutated TP53-R72 showed significantly longer survival times than those with the mutated TP53-P72 when evaluated by overall survival (p = 0.012). CONCLUSION: Using a combined mutant allele- and polymorphic allele-specific PCR, we defined the codon 72 polymorphic status of the TP53 mutated allele in Japanese CRC patients. We raised a possibility that Dukes' stage C colorectal cancer patients with tumors carrying TP53 mutation, especially the P72 allele, benefited from 5-FU based postoperative chemotherapy.

TopBP1 associates with NBS1 and is involved in homologous recombination repair.

TopBP1 is involved in DNA replication and DNA damage checkpoint. Recent studies have demonstrated that TopBP1 is a direct positive effecter of ATR. However, it is not known how TopBP1 recognizes damaged DNA. Here, we show that TopBP1 formed nuclear foci after exposure to ionizing radiation, but such TopBP1 foci were abolished in Nijmegen breakage syndrome cells. We also show that TopBP1 physically associated with NBS1 in vivo. These results suggested that NBS1 might regulate TopBP1 recruitment to the sites of DNA damage. TopBP1-depleted cells showed hypersensitivity to Mitomycin C and ionizing radiation, an increased frequency of sister-chromatid exchange level, and a reduced frequency of DNA double-strand break induced homologous recombination repair. Together, these results suggested that TopBP1 might be a mediator of DNA damage signaling from NBS1 to ATR and promote homologous recombination repair.

Heat-regulated production and secretion of insulin from a human artificial chromosome vector.

Human artificial chromosomes (HACs) behave as independent minichromosomes and are potentially useful as a way to achieve safe, long-term expression of a transgene. In this study, we sought to elucidate the potential of HAC vectors carrying the human proinsulin transgene for gene therapy of insulin-dependent diabetes mellitus (IDDM) using non-beta-cells as a host for the vector. To facilitate the production of mature insulin in non-beta-cells and to safely regulate the level of transgene expression, we introduced furin-cleavable sites into the proinsulin coding region and utilized the heat shock protein 70 (Hsp70) promoter. We used Cre-loxP-mediated recombination to introduce the gene cassettes onto 21DeltapqHAC, a HAC vector whose structure is completely defined, present in human fibrosarcoma HT1080 cells. We observed long-term expression and stable retention of the transgene without aberrant translocation of the HAC constructs. As expected, the Hsp70 promoter allowed us to regulate gene expression with temperature, and the production and secretion of intermediates of mature insulin were made possible by the furin-cleavable sites we had introduced into proinsulin. This study can be an initial step on the application of HAC vectors on the gene delivery to non-beta-cells, which might provide a direction for future treatment for diabetes.

Loss of imprinting of PEG1/MEST in lung cancer cell lines.

Paternally expressed imprinted gene 1/mesoderm-specific transcript (PEG1/MEST) is an imprinted gene expressed from the paternal allele. Recently, frequent loss of imprinting (LOI) of PEG1/MEST has been reported in lung adenocarcinomas. It is suggested that the LOI may be involved in pathogenesis of lung adenocarcinoma. In the present study, incidence of LOI of PEG1/MEST was examined in lung cancer cell lines, including small cell lung cancer (SCLC). Among 50 cell lines tested, 20 cell lines were heterozygous for the AflIII site of the PEG1/MEST gene. In these heterozygotes, biallelic expression was observed in 9 cell lines (45%), monoallelic in 11 (55%). In cell lines of non-small cell lung cancer (NSCLC), 62% (8 of 13) exhibited biallelic expression. In SCLC, only 1 of 7 cell lines (14%) showed biallelic expression. LOI of PEG1/MEST in the NSCLC cell line is significantly frequent compared with that in SCLC cell lines (p=0.043). This result supports the possibility that LOI may be related to tumorigenesis and malignant transformation, especially in NSCLC.

Use of real-time RT-PCR for the detection of allelic expression of an imprinted gene.

Measurement of the relative amounts of transcripts from two alleles is important in the study of imprinted genes, since quantitative differences that vary among tissues or individuals, and subtle differences in the ratio of allelic expression can have pathobiological significance. Discrimination of alleles is commonly based on PCR, followed by restriction endonuclease digestion to recognize a polymorphic site. However, the use of restriction enzymes misses most of the available single nucleotide polymorphisms. Practically, it requires substantial post-PCR analyses including the restriction enzyme digestion and gel electrophoresis, all of which increase turn around time. Taking advantage of our previous study identifying lung adenocarcinomas displaying biallelic expression of the imprinted gene MEST, we investigated the validity of a method of allelic discrimination in a real-time PCR assay using allele-specific probes. Allelic expression of the MEST gene in the range of 4-fold differences was detected. This new method should enhance our ability to rapidly and accurately assess allelic expression of imprinted genes in a number of samples.