Effective recovery of highly purified CD326(+) tumor cells from lavage fluid of patients treated with a novel cell-free and concentrated ascites reinfusion therapy (KM-CART).

For the production of tumor-specific vaccines, including dendritic cell (DC) vaccines, the tumor cells themselves are an ideal source. Floating tumor cells in the ascites fluid from patients with malignant ascites are a good candidate source, but it is not easy to obtain pure tumor cells from ascites because of various types of cell contamination as well as protein aggregates. We here report an effective method to recover pure tumor cells from malignant ascites. We used lavage fluid from 13 patients with malignant ascites who were treated with modified cell-free and concentrated ascites reinfusion therapy (KM-CART). Cellular components were separated from the lavage fluid by centrifugation, enzymatic digestion and hemolysis. Tumor cells were purified by depleting CD45(+) leukocytes with antibody-conjugated magnetic beads. The tumor cell lysate was extracted by freeze-and-thaw cycles. The mean obtained total cell number was 7.50 × 10(7) cells (range 4.40 × 10(6)-2.48 × 10(8) cells). From this fraction, 6.39 × 10(6) (range 3.23 × 10(5)-2.53 × 10(7)) CD45(-) cells were collected, and the tumor cell purity was over 80 % defined as CD45(-)CD326(+). A sufficient amount of tumor lysate, average  = 2416 µg (range 25-8743 µg), was extracted from CD45(-)CD326(+) tumor cells. We here established an effective method to produce highly purified tumor cells from KM-CART lavage fluid. The clinical feasibility of this simple preparation method for generating tumor lysate should be examined in clinical studies of DC vaccines.

Gemcitabine treatment enhances HER2 expression in low HER2-expressing breast cancer cells and enhances the antitumor effects of trastuzumab emtansine.

Trastuzumab emtansine (T-DM1), trastuzumab-conjugated with a cytotoxic agent, has shown promising antitumor effects in breast cancer. Since a good therapeutic response using T-DM1 treatment requires high human epidermal growth factor receptor 2 (HER2) expression, breast cancers with low or no HER2 expression have not been used for T-DM1 treatment. The aim of the present study was to show that treatment of low HER2-expressing breast cancer cells with gemcitabine (GEM) enhanced HER2 expression using RT-qPCR, immunoblot and flow cytometric analysis. The results showed that GEM treatment significantly enhanced HER2 expression in MDA-MB-231, MCF7 and BT-20 breast cancer cells, while paclitaxel (PTX) treatment induced lower or no enhancement in HER2 expression. The expression of HER2 mRNA was also enhanced in GEM-treated MCF7 cells. Treatment with an inhibitor for nuclear factor-(NF)-κB suppressed GEM-induced HER2 upregulation, indicating that NF-κB activation by GEM may be associated with HER2 upregulation. T-DM1 binding to HER2 on MCF-7 cells was enhanced by GEM pretreatment and the combined treatment of GEM and T-DM1 synergistically inhibited the proliferation of MCF7 cells. Thus, the combined treatment with GEM and T-DM1 may be a promising therapeutic modality for low HER2-expressing breast cancers, which was facilitated by the unique HER2-upregulating effect of GEM.

Treatment with chemotherapy and dendritic cells pulsed with multiple Wilms' tumor 1 (WT1)-specific MHC class I/II-restricted epitopes for pancreatic cancer.

PURPOSE: We performed a phase I trial to investigate the safety, clinical responses, and Wilms' tumor 1 (WT1)-specific immune responses following treatment with dendritic cells (DC) pulsed with a mixture of three types of WT1 peptides, including both MHC class I and II-restricted epitopes, in combination with chemotherapy. EXPERIMENTAL DESIGN: Ten stage IV patients with pancreatic ductal adenocarcinoma (PDA) and 1 patient with intrahepatic cholangiocarcinoma (ICC) who were HLA-positive for A*02:01, A*02:06, A*24:02, DRB1*04:05, DRB1*08:03, DRB1*15:01, DRB1*15:02, DPB1*05:01, or DPB1*09:01 were enrolled. The patients received one course of gemcitabine followed by biweekly intradermal vaccinations with mature DCs pulsed with MHC class I (DC/WT1-I; 2 PDA and 1 ICC), II (DC/WT1-II; 1 PDA), or I/II-restricted WT1 peptides (DC/WT1-I/II; 7 PDA), and gemcitabine. RESULTS: The combination therapy was well tolerated. WT1-specific IFNγ-producing CD4(+) T cells were significantly increased following treatment with DC/WT1-I/II. WT1 peptide-specific delayed-type hypersensitivity (DTH) was detected in 4 of the 7 patients with PDA vaccinated with DC/WT1-I/II and in 0 of the 3 patients with PDA vaccinated with DC/WT1-I or DC/WT1-II. The WT1-specific DTH-positive patients showed significantly improved overall survival (OS) and progression-free survival (PFS) compared with the negative control patients. In particular, all 3 patients with PDA with strong DTH reactions had a median OS of 717 days. CONCLUSIONS: The activation of WT1-specific immune responses by DC/WT1-I/II combined with chemotherapy may be associated with disease stability in advanced pancreatic cancer.

Prognostic factors related to add-on dendritic cell vaccines on patients with inoperable pancreatic cancer receiving chemotherapy: a multicenter analysis.

OBJECTIVE: Dendritic cell (DC)-based cancer vaccines may have a significant benefit to patients with advanced pancreatic cancer. However, variations among clinical studies make it difficult to compare clinical outcomes. Here, we identified factors that determined the clinical benefits by analyzing data obtained at seven Japanese institutions that employed the same DC preparation and treatment regimens. METHODS: Of 354 patients who met the inclusion criteria, 255 patients who received standard chemotherapy combined with peptide-pulsed DC vaccines were analyzed. RESULTS: The mean survival time from diagnosis was 16.5 months (95 % CI 14.4-18.5) and that from the first vaccination was 9.9 months (95 % CI 8.0-12.9). Known prognostic baseline factors related to advanced pancreatic cancer, namely ECOG-PS, peritoneal metastasis, liver metastasis, and the prognostic nutrition index, were also representative. Importantly, we found that erythema reaction after vaccination was an independent and treatment-related prognostic factor for better survival and that OK-432 might be a good adjuvant enhancing the antitumor immunity during DC vaccination. CONCLUSIONS: This is the first report of a multicenter clinical study suggesting the feasibility and possible clinical benefit of an add-on DC vaccine in patients with advanced pancreatic cancer who are undergoing chemotherapy. These findings need to be addressed in well-controlled prospective randomized trials.

Impact of dendritic cell vaccines pulsed with Wilms' tumour-1 peptide antigen on the survival of patients with advanced non-small cell lung cancers.

PURPOSE: Dendritic cell (DC)-based vaccines have been expected to serve as new therapeutic approaches for advanced non-small cell lung cancers (NSCLCs); however, their clinical outcomes have not been fully elucidated. We report a single-centre clinical study analysing factors affecting the survival of patients with advanced NSCLCs who received DC vaccines pulsed with or without Wilms' tumour protein-1 (WT1) peptide. METHODS: Among 62 patients with previously treated inoperable or postoperatively relapsed NSCLCs who met the inclusion criteria, DCs from 47 (76%) patients who showed HLA-A2402/0201/0206 were pulsed with one or more corresponding WT1 peptide antigens. DC vaccines were intradermally injected biweekly. RESULTS: Clinical responses based on response evaluation criteria in solid tumours (RECIST) were found in 31 (50%) patients at 3 months after the first DC vaccine (complete response: 1 (1.6%), partial response: 4 (6.5%), stable disease: 26 (41.9%)). Median survival time was 27 months (82% in 1 year and 54% in 2 years) from initial diagnosis, and that was 12 months (48% in 1 year and 22% in 2 years) from the first DC vaccination. Importantly, multivariate analyses revealed that only two factors, blood haemoglobin and the use of WT1 peptides, significantly affected the overall survival of patients from both initial diagnosis and first vaccination. CONCLUSIONS: This study is the first to suggest that DC vaccines pulsed with WT1 may hold a significant impact to prolong the overall survival of patients with advanced NSCLCs.

One-step detection of the 2009 pandemic influenza A(H1N1) virus by the RT-SmartAmp assay and its clinical validation.

BACKGROUND: In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society. METHODOLOGY: To address the clinical need for rapid diagnosis, we have developed a new method, the "RT-SmartAmp assay", to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses. RESULTS AND CONCLUSIONS: We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.

Association between breast cancer risk and the wild-type allele of human ABC transporter ABCC11.

BACKGROUND: International mortality and frequency rates for breast cancer have been associated with the wet type of human earwax. It was recently found that earwax type is determined by a single nucleotide polymorphism (SNP), 538G>A (Gly180Arg), in ABCC11. The G allele determines the wet type of earwax as a Mendelian trait with a dominant phenotype. The present study examined the association between the frequency rate of breast cancer and the frequency of the G allele of ABCC11. PATIENTS AND METHODS: Using blood samples from patients with invasive breast cancer (n = 270) and control volunteers (n = 273), the 538G>A SNP in ABCC11 was genotyped using the SmartAmp method. RESULTS: The frequency of the G allele in breast cancer patients was higher than that in healthy controls. The odds ratio for the genotypes (G/G+G/A) to develop breast cancer was estimated to be 1.63 (p-value = 0.026), suggesting that the G allele in ABCC11 is associated with breast cancer risk. CONCLUSION: This study showed that Japanese women with wet earwax have a higher relative risk of developing breast cancer than those with dry earwax. The ABCC11 SNPs that determine these phenotypes should be further investigated in order to obtain insights into the mechanisms by which breast cancer develops and progresses.

Exciton Primer-mediated SNP detection in SmartAmp2 reactions.

Most commonly used intercalating fluorescent dyes in DNA detection are lacking any sequence specificity, whereas so-called Exciton Primers can overcome this limitation by functioning as "sequence-specific dyes." After hybridization to complementary sequences, the fluorescence of Exciton Primers provides sequence-specific signals for real-time monitoring of amplification reactions. Applied to the SmartAmp2 mutation detection process, Exciton Primers show high signal strength with low background leading to a superior specificity and sensitivity compared to SYBR Green I. Signal strength can be further enhanced using multiple dyes within one Exciton Primer or use of multiple Exciton Primers in the same amplification reaction. Here we demonstrate the use of Exciton Primers for genotyping a single nucleotide polymorphism (SNP) in the VKORC1 locus (-1639G>A) relevant for Warfarin dosing as an example for Exciton Primers mediated genotyping by SmartAmp2. The genotyping assay can use only one labeled Exciton Primer for endpoint detection, or simultaneously by real-time monitoring detect wild-type and mutant alleles in a one-tube reaction using two Exciton Primers having different dyes. Working directly from blood samples, Exciton Primer mediated genotyping by SmartAmp2 offers superior solutions for rapid point-of-care testing.

Usefulness of peptide nucleic acid (PNA)-clamp smart amplification process version 2 (SmartAmp2) for clinical diagnosis of KRAS codon 12 mutations in lung adenocarcinoma: comparison of PNA-clamp SmartAmp2 and PCR-related methods.

KRAS is an oncogene that can be activated by mutations. Patients with non-small cell lung cancer who have KRAS mutations do not respond to tyrosine kinase inhibitors; therefore, accurate detection of KRAS mutations is important for deciding therapeutic strategies. Although sequencing-related techniques have been frequently used, they are usually too complex, have low sensitivity, and are time-consuming for routine screening in clinical situations. We evaluated peptide nucleic acid (PNA)-clamp smart amplification process version 2 (SmartAmp2) as a detection method for KRAS codon 12 mutations in patient specimens compared with traditional sequencing and polymerase chain reaction-related methods. Among 172 lung adenocarcinoma samples, direct sequencing, enzyme-enriched sequencing, and PNA-enriched sequencing showed that 16 (9.3%), 26 (15.7%), and 28 (16.3%) tumors, respectively, contained KRAS mutations in codon 12. Using PNA-clamp SmartAmp2, we could identify 31 (18.0%) tumors that had KRAS mutations in codon 12 within 60 minutes, three of which were undetected by polymerase chain reaction-related methods. On the other hand, we examined 30 nonmalignant peripheral lung tissue specimens and found no mutations in any of the samples using PNA-clamp SmartAmp2. In this study, we confirmed that PNA-clamp SmartAmp2 has high sensitivity and accuracy and is suitable for the clinical diagnosis of KRAS codon 12 mutations.

Isothermal single nucleotide polymorphism genotyping and direct PCR from whole blood using a novel whole-blood lysis buffer.

Cell lysis and subsequent release of genomic DNA is an ongoing dilemma for molecular biological techniques. In most cases, technologies such as PCR and other amplification techniques require DNA extraction and purification steps. The Smart Amplification Process Version 2 (SmartAmp2) is an isothermal and integrated amplification technology that eliminates the need for time-consuming sample preparation for the rapid detection of nucleic acids, including single nucleotide polymorphisms (SNPs), mutations, and other targets. In addition, DNA amplification directly from whole blood is beneficial and lessens the risk of cross-contamination. Traditional SmartAmp2 assays entail two steps and require an alkali pretreatment step at 98 degrees C prior to the 60 degrees C run. To make SmartAmp2 truly isothermal and to simplify DNA amplification, we hereby introduce the SmartAmp Isothermal Lysis Buffer (SIL-B), a newly developed chaotropic lysis buffer that enables the simultaneous recovery and denaturation of genomic material directly from whole blood at a uniform 60 degrees C. The improved method for isolating nucleic acids from whole blood is a critical milestone in making SmartAmp2 truly isothermal from start to finish at one temperature, increasing its potential to be routinely used in field point-of-care testing. Furthermore, pretreatment with SIL-B enables the PCR amplification of genomic material directly from whole blood.

Rapid single-nucleotide polymorphism detection of cytochrome P450 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genes for the warfarin dose adjustment by the SMart-amplification process version 2.

BACKGROUND: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. METHODS: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. RESULTS: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. CONCLUSIONS: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.

Rapid screening assay for KRAS mutations by the modified smart amplification process.

Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.

A B-Myb complex containing clathrin and filamin is required for mitotic spindle function.

B-Myb is one member of the vertebrate Myb family of transcription factors and is ubiquitously expressed. B-Myb activates transcription of a group of genes required for the G2/M cell cycle transition by forming the dREAM/Myb-MuvB-like complex, which was originally identified in Drosophila. Mutants of zebrafish B-myb and Drosophila myb exhibit defects in cell cycle progression and genome instability. Although the genome instability caused by a loss of B-Myb has been speculated to be due to abnormal cell cycle progression, the precise mechanism remains unknown. Here, we have purified a B-Myb complex containing clathrin and filamin (Myb-Clafi complex). This complex is required for normal localization of clathrin at the mitotic spindle, which was previously reported to stabilize kinetochore fibres. The Myb-Clafi complex is not tightly associated with the mitotic spindles, suggesting that this complex ferries clathrin to the mitotic spindles. Thus, identification of the Myb-Clafi complex reveals a previously unrecognized function of B-Myb that may contribute to its role in chromosome stability, possibly, tumour suppression.

Abnormal development of urogenital organs in Dlgh1-deficient mice.

Dlgh1 (discs large homolog 1) is a mammalian homolog of the Drosophila tumor suppressor Discs large 1, and is a member of the membrane-associated guanylate kinase (MAGUK) scaffolding proteins that contain three PSD-95/Dlg/ZO-1 (PDZ) domains. Discs large 1 is involved in epithelial polarization and cell-cell adhesion complex formation during Drosophila development. However, the functions of Dlgh1 during mammalian development remain to be elucidated. We generated Dlgh1-knockout mice and found that homozygous Dlgh1-knockout mice developed various abnormalities in their renal and urogenital organs. The kidneys and ureters were hypoplastic and the lower ends of the ureters were ectopic. In addition, the vagina and seminal vesicle, which are derived from the lower part of the Müllerian and Wolffian duct, respectively, were absent. Unexpectedly, loss of Dlgh1 function in the developing ureters did not disrupt cell-cell junctional complexes, but did impair cellular proliferation in the epithelium. These results suggest a novel role for Dlgh1 in regulating epithelial duct formation and morphogenesis during mammalian development. Although congenital absence of the vagina associated with other variable Müllerian duct abnormalities has been reported in humans, its mechanism has not yet been clarified. Our findings might contribute to a better understanding of such abnormalities.

The mammalian homolog of the Drosophila discs large tumor suppressor protein up-regulates expression of the ELR+ CXC chemokine Scyb5.

The mammalian homolog of the Drosophila discs large tumor suppressor protein Dlg functions as a scaffolding protein that facilitates the transmission of diverse signals. In the present study, we attempted to identify the downstream target genes of Dlg, and found that Dlg up-regulates expression of the ELR+ CXC chemokine Scyb5, which has been implicated in the immune system. Our finding suggests that Scyb5 may play an important role in the tumor suppressor function of Dlg.

Anteriorization of neural fate by inhibitor of beta-catenin and T cell factor (ICAT), a negative regulator of Wnt signaling.

Inhibitor of beta-catenin and T cell factor (ICAT) inhibits Wnt signaling by interfering with the interaction between beta-catenin and T cell factor. Here we show that ICAT(-/-) embryos exhibit malformation of the forebrain and craniofacial bones and lack the kidney. Analysis of the neuronal differentiation of embryonic stem cells revealed that Wnt3a redirects the fate of neural progenitors to a posterior character, whereas ICAT induces forebrain cells by inhibiting Wnt signaling. Furthermore, ICAT(-/-) embryonic stem cells were found to differentiate into neuronal cells possessing a posterior character. These results suggest that ICAT plays an important role in the anteriorization of neural cells by inhibiting the posteriorizing activity of Wnt signaling.

Binding of the mammalian homolog of the Drosophila discs large tumor suppressor protein to the ribosome receptor.

DLG, the mammalian homolog of the Drosophila Discs Large suppressor protein, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. In the present study, we attempted to identify partner proteins for DLG, and found that DLG interacts through its PDZ domains with the ribosome receptor. The ribosome receptor is an integral endoplasmic reticulum protein that has been suggested to be involved in secretion. Our finding raises the possibility that DLG plays a role in the regulation of secretion by interacting with the ribosome receptor.