Retrospective, Multicenter Analysis Comparing Conventional with Oncoplastic Breast Conserving Surgery: Oncological and Surgical Outcomes in Women with High-Risk Breast Cancer from the OPBC-01/iTOP2 Study.

INTRODUCTION: Recent data suggest that margins ≥2 mm after breast-conserving surgery may improve local control in invasive breast cancer (BC). By allowing large resection volumes, oncoplastic breast-conserving surgery (OBCII; Clough level II/Tübingen 5-6) may achieve better local control than conventional breast conserving surgery (BCS; Tübingen 1-2) or oncoplastic breast conservation with low resection volumes (OBCI; Clough level I/Tübingen 3-4). METHODS: Data from consecutive high-risk BC patients treated in 15 centers from the Oncoplastic Breast Consortium (OPBC) network, between January 2010 and December 2013, were retrospectively reviewed. RESULTS: A total of 3,177 women were included, 30% of whom were treated with OBC (OBCI n = 663; OBCII n = 297). The BCS/OBCI group had significantly smaller tumors and smaller resection margins compared with OBCII (pT1: 50% vs. 37%, p = 0.002; proportion with margin <1 mm: 17% vs. 6%, p < 0.001). There were significantly more re-excisions due to R1 ("ink on tumor") in the BCS/OBCI compared with the OBCII group (11% vs. 7%, p = 0.049). Univariate and multivariable regression analysis adjusted for tumor biology, tumor size, radiotherapy, and systemic treatment demonstrated no differences in local, regional, or distant recurrence-free or overall survival between the two groups. CONCLUSIONS: Large resection volumes in oncoplastic surgery increases the distance from cancer cells to the margin of the specimen and reduces reexcision rates significantly. With OBCII larger tumors are resected with similar local, regional and distant recurrence-free as well as overall survival rates as BCS/OBCI.

Absence of Islet Autoantibodies and Modestly Raised Glucose Values at Diabetes Diagnosis Should Lead to Testing for MODY: Lessons From a 5-Year Pediatric Swedish National Cohort Study.

OBJECTIVE: Identifying maturity-onset diabetes of the young (MODY) in pediatric populations close to diabetes diagnosis is difficult. Misdiagnosis and unnecessary insulin treatment are common. We aimed to identify the discriminatory clinical features at diabetes diagnosis of patients with glucokinase (GCK), hepatocyte nuclear factor-1A (HNF1A), and HNF4A MODY in the pediatric population. RESEARCH DESIGN AND METHODS: Swedish patients (n = 3,933) aged 1-18 years, diagnosed with diabetes May 2005 to December 2010, were recruited from the national consecutive prospective cohort Better Diabetes Diagnosis. Clinical data, islet autoantibodies (GAD insulinoma antigen-2, zinc transporter 8, and insulin autoantibodies), HLA type, and C-peptide were collected at diagnosis. MODY was identified by sequencing GCK, HNF1A, and HNF4A, through either routine clinical or research testing. RESULTS: The minimal prevalence of MODY was 1.2%. Discriminatory factors for MODY at diagnosis included four islet autoantibody negativity (100% vs. 11% not-known MODY; P = 2 × 10-44), HbA1c (7.0% vs. 10.7% [53 vs. 93 mmol/mol]; P = 1 × 10-20), plasma glucose (11.7 vs. 26.7 mmol/L; P = 3 × 10-19), parental diabetes (63% vs. 12%; P = 1 × 10-15), and diabetic ketoacidosis (0% vs. 15%; P = 0.001). Testing 303 autoantibody-negative patients identified 46 patients with MODY (detection rate 15%). Limiting testing to the 73 islet autoantibody-negative patients with HbA1c <7.5% (58 mmol/mol) at diagnosis identified 36 out of 46 (78%) patients with MODY (detection rate 49%). On follow-up, the 46 patients with MODY had excellent glycemic control, with an HbA1c of 6.4% (47 mmol/mol), with 42 out of 46 (91%) patients not on insulin treatment. CONCLUSIONS: At diagnosis of pediatric diabetes, absence of all islet autoantibodies and modest hyperglycemia (HbA1c <7.5% [58 mmol/mol]) should result in testing for GCK, HNF1A, and HNF4A MODY. Testing all 12% patients negative for four islet autoantibodies is an effective strategy for not missing MODY but will result in a lower detection rate. Identifying MODY results in excellent long-term glycemic control without insulin.

Decrease by 50% of plasma IgA tissue transglutaminase antibody concentrations within 2 months after start of gluten-free diet in children with celiac disease used as a confirming diagnostic test.

BACKGROUND: Histological examination of small bowel biopsies is normally the gold standard for the diagnosis of celiac disease (CD). The objective of this study was to investigate whether the rate of decreases in elevated plasma IgA tissue transglutaminase antibody (IgA-tTG) and/or IgG deamidated gliadin peptides antibody (IgG - DGP) concentrations could be used as a confirming test for CD in children on a gluten-free diet (GFD) when biopsy was omitted in the diagnostic process. METHODS: In this retrospective study we compared children (≤18 years old) with a CD-confirming biopsy (n = 16) to children without a biopsy (n = 18). After initiation of GFD the antibody half-life (the time (T½) when the antibody concentration is 50% decreased) was determined in all children. RESULTS: Children with a biopsy (IgA-tTG, T½ = 1.9 months; IgG - DGP, T½ = 2.2 months) and children without a biopsy (IgA-tTG, T½ = 1.6 months; IgG - DGP, T½ = 2.7 months) had comparable T½ (mean) results (p < 0.05) supporting that all children had the CD diagnosis. CONCLUSIONS: When biopsy was omitted a rapid rate of decrease in CD antibody concentrations confirmed the CD diagnosis in children on GFD. The half-lives (T½) of IgA-tTG were less than 2 months in CD children.

Levels of beta-microseminoprotein in blood and risk of prostate cancer in multiple populations.

BACKGROUND: A common genetic variant (rs10993994) in the 5' region of the gene encoding ß-microseminoprotein (MSP) is associated with circulating levels of MSP and prostate cancer risk. Whether MSP levels are predictive of prostate cancer risk has not been evaluated. METHODS: We investigated the prospective relationship between circulating plasma levels of MSP and prostate cancer risk in a nested case-control study of 1503 case subjects and 1503 control subjects among black, Latino, Japanese, Native Hawaiian, and white men from the Multiethnic Cohort study. We also examined the ability of MSP to serve as a biomarker for discriminating prostate cancer case subjects from control subjects. All statistical tests are two-sided. RESULTS: In all racial and ethnic groups, men with lower MSP levels were at greater risk of developing prostate cancer (odds ratio = 1.02 per one unit decrease in MSP, P < .001 in the prostate-specific antigen [PSA]-adjusted analysis). Compared with men in the highest decile of MSP, the multivariable PSA-adjusted odds ratio was 3.64 (95% confidence interval = 2.41 to 5.49) for men in the lowest decile. The positive association with lower MSP levels was observed consistently across racial and ethnic populations, by disease stage and Gleason score, for men with both high and low levels of PSA and across all genotype classes of rs10993994. However, we did not detect strong evidence of MSP levels in improving prostate cancer prediction beyond that of PSA. CONCLUSIONS: Regardless of race and ethnicity or rs10993994 genotype, men with low blood levels of MSP have increased risk of prostate cancer.

Polymorphisms at the Microseminoprotein-beta locus associated with physiologic variation in beta-microseminoprotein and prostate-specific antigen levels.

BACKGROUND: rs10993994, a single nucleotide polymorphism (SNP) at the genetic locus encoding beta-microseminoprotein (beta-MSP), is associated with both prostate cancer risk and levels of blood prostate-specific antigen (PSA), a biomarker used in prostate cancer screening. Therefore, we wished to determine the association between SNPs at MSMB, the gene encoding beta-MSP, and the levels of prostate-produced biomarkers beta-MSP, PSA, and human kallikrein 2 (hK2) in blood and semen. METHODS: Blood and semen from 304 healthy young Swedish men (ages 18-21) were assayed for beta-MSP, PSA, and hK2. SNPs around MSMB were genotyped from matched DNA and analyzed for quantitative association with biomarker levels. Empirical P values were multiple test-corrected and the independence of each SNP's effect was determined. RESULTS: rs10993994 was significantly associated with the blood and semen levels of beta-MSP (both P < 1.0 x 10(-7)) and PSA (P = 0.00014 and P = 0.0019), and semen levels of hK2 (P = 0.00027). Additional copies of the prostate cancer risk allele resulted in lower beta-MSP but higher PSA levels, and singly explained 23% and 5% of the variation seen in semen beta-MSP and PSA, respectively. Additional SNPs at MSMB are associated with beta-MSP and PSA independently of rs10993994. CONCLUSIONS: SNPs at MSMB correlate with physiologic variation in beta-MSP and PSA levels in the blood and semen of healthy young Swedish men. In particular, rs10993994 has a strong effect on beta-MSP levels. IMPACT: Our results suggest a mechanism by which rs10993994 might predispose to prostate cancer and raise the possibility that genetic variation might need to be considered in interpreting the levels of these biomarkers.

A common prostate cancer risk variant 5' of microseminoprotein-beta (MSMB) is a strong predictor of circulating beta-microseminoprotein (MSP) levels in multiple populations.

BACKGROUND: ß-Microseminoprotein (MSP) is one of the three most abundantly secreted proteins of the prostate and has been suggested as a biomarker for prostate cancer risk. A common variant, rs10993994, in the 5' region of the gene that encodes MSP (MSMB) has recently been identified as a risk factor for prostate cancer. METHODS: We examined the association between rs10993994 genotype and MSP levels in a sample of 500 prostate cancer-free men from four racial/ethnic populations in the Multiethnic Cohort (European Americans, African Americans, Latinos, and Japanese Americans). Generalized linear models were used to estimate the association between rs10993994 genotype and MSP levels. RESULTS: We observed robust associations between rs10994994 genotype and MSP levels in each racial/ethnic population (all P < 10(-8)), with carriers of the C allele having lower geometric mean MSP levels (ng/mL; CC/CT/TT genotypes: European Americans, 28.8/20.9/10.0; African Americans, 29.0/21.9/10.9; Latinos, 29.2/17.1/8.3; and Japanese Americans, 25.8/16.4/6.7). We estimated the variant accounts for 30% to 50% of the variation in MSP levels in each population. We also observed significant differences in MSP levels between populations (P = 3.5 × 10(-6)), with MSP levels observed to be highest in African Americans and lowest in Japanese Americans. CONCLUSIONS: Rs10993994 genotype is strongly associated with plasma MSP levels in multiple racial/ethnic populations. IMPACT: This supports the hypothesis that rs10993994 may be the biologically functional allele.

Rapidly evolving marmoset MSMB genes are differently expressed in the male genital tract.

BACKGROUND: Beta-microseminoprotein, an abundant component in prostatic fluid, is encoded by the potential tumor suppressor gene MSMB. Some New World monkeys carry several copies of this gene, in contrast to most mammals, including humans, which have one only. Here we have investigated the background for the species difference by analyzing the chromosomal organization and expression of MSMB in the common marmoset (Callithrix jacchus). METHODS: Genes were identified in the Callithrix jacchus genome database using bioinformatics and transcripts were analyzed by RT-PCR and quantified by real time PCR in the presence of SYBR green. RESULTS: The common marmoset has five MSMB: one processed pseudogene and four functional genes. The latter encompass homologous genomic regions of 32-35 kb, containing the genes of 12-14 kb and conserved upstream and downstream regions of 14-19 kb and 3-4 kb. One gene, MSMB1, occupies the same position on the chromosome as the single human gene. On the same chromosome, but several Mb away, is another MSMB locus situated with MSMB2, MSMB3 and MSMB4 arranged in tandem. Measurements of transcripts demonstrated that all functional genes are expressed in the male genital tract, generating very high transcript levels in the prostate. The transcript levels in seminal vesicles and testis are two and four orders of magnitude lower. A single gene, MSMB3, accounts for more than 90% of MSMB transcripts in both the prostate and the seminal vesicles, whereas in the testis around half of the transcripts originate from MSMB2. These genes display rapid evolution with a skewed distribution of mutated nucleotides; in MSMB2 they affect nucleotides encoding the N-terminal Greek key domain, whereas in MSMB3 it is the C-terminal MSMB-unique domain that is affected. CONCLUSION: Callitrichide monkeys have four functional MSMB that are all expressed in the male genital tract, but the product from one gene, MSMB3, will predominate in seminal plasma. This gene and MSMB2, the predominating testicular gene, have accumulated mutations that affect different parts of the translation products, suggesting an ongoing molecular specialization that presumably yields functional differences in accessory sex glands and testis.

Beta-microseminoprotein in serum correlates with the levels in seminal plasma of young, healthy males.

Beta-microseminoprotein (MSP) is one of the most abundant proteins secreted by the prostate gland. Because MSP is also synthesized in nonreproductive organs, the establishment of a solid relationship between the levels of MSP in serum and semen is crucial for future studies connecting MSP with aging or diseases of the prostate gland. We developed a specific, competitive, europium-based immunoassay to measure MSP in serum and seminal plasma. We also produced recombinant MSP in insect cells using baculo virus and purified it to homogeneity by a novel approach with ethanol extraction and gel filtration. The median values of MSP in 205 young men were 12 microg/L (2.5-97.5 percentile, 4.9-26 microg/L) in serum and 0.53 g/L (2.5-97.5 percentile, 0.13-2.0 g/L) or 1.8 mg (2.5-97.5 percentile, 0.32-6.6 mg) in seminal plasma. MSP in serum showed significant correlation to MSP in seminal plasma (r = .50, P < .001). Significant correlations were also found in seminal plasma between MSP and prostate-specific antigen (PSA) (r = .65, P < .001) and between MSP and Zn(2+) (r = .54, P < .001). The yield of recombinant MSP in culture medium was 35 mg/L or higher, and recovery following ethanol extraction was 80%-90%. MSP in serum reflects the prostate secretion of MSP, and correlations were also found in seminal plasma between MSP and PSA and Zn(2+). This suggests that MSP in serum can be used as a marker of prostate secretion, despite the contribution from extra prostatic tissues.

The cotton-top tamarin (Saguinus oedipus) has five beta-microseminoprotein genes, two of which are pseudogenes.

beta-Microseminoprotein (MSP) is one of the most abundant proteins in human seminal plasma and is secreted from the prostate gland. Its evolution can be traced from primates down to nonvertebrate species such as amphioxus, despite substantial differences in the primary structure. Most mammals are known to have one single MSP gene, but we have previously shown that the cotton-top tamarin and the common marmoset-two New World monkeys-carry several MSP genes. In this study we continue our characterization of MSP genes in the cotton-top tamarin by presenting the full nucleotide sequence of the three previously identified genes, mspA, mspE, and mspJ. A promoter analysis using the luciferase reporter showed that mspE is as transcriptionally active as the single human MSP gene, whereas mspA and mspJ display no activity with this assay. Two novel MSP genes were also identified, mspB and mspH, both of which are pseudogenes. MspB has a frameshift mutation in the third exon resulting in a new C-terminus and premature stop of translation. MspH has the features of a processed pseudogene, originating from a transcript of mspE. It is integrated into the genome together with another processed pseudogene originating from a transcript of the nucleoporin gene NUP88. The MSP genes described in this study probably arose by phylogenetically rather late duplication or retrotransposition, suggesting that they are confined to a limited number of New World monkeys.

A highly conserved protein secreted by the prostate cancer cell line PC-3 is expressed in benign and malignant prostate tissue.

In this study we characterize a novel gene on human chromosome 9 and its translation product, PC3-secreted microprotein (PSMP). The gene contains three exons that encode a protein of 139 amino acid residues, including a predicted signal peptide of 36 residues. The molecule is homologous to beta-microseminoprotein (MSP), a protein of unknown function, secreted at high concentration by the prostate gland. These two proteins have only 23% sequence identity, but their common origin is revealed by a preserved pattern of Cys residues. In contrast to MSP, which shows poor conservation between species, PSMP is very conserved. High transcript levels were detected in the prostate cancer cell line PC-3. Antiserum raised against PSMP detected a protein with an apparent molecular mass of 18 kDa in culture medium conditioned by PC-3 cells, but in cell lysates the antiserum also recognized a molecular species of 16 kDa, suggesting that PSMP undergoes post-translational modification. Xenografted PC-3 cell tumors in athymic nude mice showed strong staining for both PSMP protein and mRNA. Studies on human prostate cancer specimens showed immunohistochemical staining of both tumor and benign glandular cells. Our results suggest that PSMP is an important protein with significance in prostate cancer.

The common marmoset (Callithrix jacchus) has two very similar semenogelin genes as the result of gene conversion.

The semen coagulum proteins have undergone substantial structural changes during evolution. In primates, these seminal vesicle-secreted proteins are known as semenogelin I (SEMG1) and semenogelin II (SEMG2). Previous studies on the common marmoset (Callithrix jacchus) showed that ejaculated semen from this New World monkey contains semenogelin, but it remained unclear whether it carries both genes or only SEMG1 and no SEMG2, like the closely related cotton-top tamarin (Saguinus oedipus). In this study we show that there are two genes, both expressed in the seminal vesicles. Surprisingly, the genes show an almost perfect sequence identity in a region of 1.25 kb, encompassing nearly half of the genes and containing exon 1, intron 1, and the first 0.9 kb of exon 2. The underlying molecular mechanism is most likely gene conversion, and a phylogenetic analysis suggests that SEMG1 is the most probable donor gene. The marmoset SEMG1 in this report differs from a previously reported cDNA by a lack of nucleotides encoding one repeat of 60 amino acids, suggesting that marmoset SEMG1 displays allelic size variation. This is similar to what was recently demonstrated in humans, but in marmosets the polymorphism was generated by a repeat duplication, whereas in humans it was a deletion. Together, these studies shed new light on the evolution of semenogelins and the mechanisms that have generated the structural diversity of semen coagulum proteins.

beta-Microseminoprotein binds CRISP-3 in human seminal plasma.

beta-Microseminoprotein (MSP) and cysteine-rich secretory protein 3 (CRISP-3) are abundant constituents of human seminal plasma. Immunoprecipitation and gel filtration of seminal plasma proteins combined with examination of the proteins in their pure form showed that MSP and CRISP-3 form stable, non-covalent complexes. CRISP-3 binds MSP with very high affinity, as evidenced by surface plasmon resonance. Due to far higher abundance of MSP in prostatic fluid, it manifests large overcapacity for CRISP-3 binding. Structural similarity with an MSP-binding protein from blood plasma suggests that CRISP-3 binds MSP through its aminoterminal SCP-domain.

Ejaculates from the common marmoset (Callithrix jacchus) contain semenogelin and beta-microseminoprotein but not prostate-specific antigen.

Human seminal plasma contains high concentrations of prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), beta-microseminoprotein (MSP), semenogelin I (SgI), and semenogelin II (SgII), whereas only PAP and MSP are present in rodents. In order to gain a better understanding of the evolution and function of semen proteins, we have studied ejaculates from the common marmoset (Callithrix jacchus)-a New World monkey. Semen samples were analyzed with SDS-PAGE, Western blotting, and isoelectric focusing. Under reducing conditions the dominating protein components appear as heterogeneous material of 55-70 kDa and distinct protein bands of 85, 17, 16, and 15 kDa. The heterogeneous material contains glycosylated material detected by an antiserum recognizing both human SgI and SgII. Southern blotting indicates that the common marmoset has genes for both SgI and SgII. There are several marmoset MSP genes, but the strong immunoreactivity against one 15 kDa semen component with pI 7.3 suggests preferential expression of one gene in the prostate. Expression of two other genes cannot be excluded as indicated by weak reaction to isoforms with pI 6.6 and 4.9. Unexpectedly, PSA was not detected by either immunological methods or activity measurements. This is in agreement with results from Southern blotting suggesting that the common marmoset might not have a PSA gene. Thus, in this study we have shown that semen coagulum proteins are present in marmoset seminal plasma, but the lack of PSA precludes a similar liquefaction as of human semen.

The evolution of the glandular kallikrein locus: identification of orthologs and pseudogenes in the cotton-top tamarin.

Comparisons of the glandular kallikreins loci in human, mouse and rat revealed remarkable differences. For example, the mouse and the rat lack the genes encoding prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). In contrast, the intergenic region between KLK1 and KLK15 is devoid of genes and spans only 1.5 kb in humans, but encompasses 23 KLK1-like genes spanning 290 kb in the mouse. To further elucidate the evolution of glandular kallikrein genes, we investigated the structure and organization of these genes in the cotton-top tamarin (Saguinus oedipus), a New World monkey. We conclude that this species has no PSA gene. Moreover, the ortholog of the hK2 gene is a pseudogene, as it contains several mutations that preclude formation of a functional serine protease. The expression of this gene was probably silenced by a 15-bp deletion observed in an androgen response element in the upstream promoter region. Replacing the deleted base pairs in vitro with nucleotides from the human counterpart dramatically restored the transcriptional activity to a level that even surpassed that of the human ortholog. We also determined the nucleotide sequence of KLK15 and the intergenic region between this gene and KLK1 in the cotton-top tamarin. The region between KLK1 and KLK15 is conserved between the cotton-top tamarin and humans, and there are no signs of the extension seen in the mouse. KLK15 appeared to be functional, thus, we predict that it generates a protease with specificity similar to that of the human ortholog.

Molecular cloning of complementary DNA encoding mouse seminal vesicle-secreted protein SVS I and demonstration of homology with copper amine oxidases.

The primary structure of mouse SVS I was determined by peptide sequencing and nucleotide sequencing of cloned cDNA. The precursor molecule consists of 820 amino acid residues, including a signal peptide of 24 residues, and the mature polypeptide chain of 91 kDa has one site for potential N-linked glycosylation. The SVS I is homologous with amiloride-binding protein 1 (ABP1), a diamine oxidase. However, it probably lacks enzymatic activity, because the cDNA codes for His instead of Tyr at the position of the active-site topaquinon. The SVS I monomer probably binds one molecule of copper, because the His residues coordinated by Cu(II) are conserved. The SVS I gene consists of five exons and is situated on mouse chromosome 6,B2.3. It is located in a region of 100 kilobases (kb) containing several genes with homology to SVS I, including the gene of ABP1 and two other proteins with homology to diamine oxidase. The locus is conserved on rat chromosome 4q24, but the homologous region on human chromosome 7q34-q36 solely contains ABP1. The other genes with homology to diamine oxidase were probably present in a progenitor of primates and rodents but were lost in the evolutionary lineage leading to humans-presumably during recombination between chromosomes. The estimated molecular mass of rat SVS I is 102 kDa (excluding glycosylation). The species difference in size of SVS I is caused by tandem repeats of 18 amino acid residues in the central part of the molecule: The mouse has seven repeats, and the rat has 12 repeats.