Tumor Content Chart-Assisted HER2/CEP17 Digital PCR Analysis of Gastric Cancer Biopsy Specimens.

Evaluating HER2 gene amplification is an essential component of therapeutic decision-making for advanced or metastatic gastric cancer. A simple method that is applicable to small, formalin-fixed, paraffin-embedded biopsy specimens is desirable as an adjunct to or as a substitute for currently used HER2 immunohistochemistry and in situ hybridization protocols. In this study, we developed a microfluidics-based digital PCR method for determining HER2 and chromosome 17 centromere (CEP17) copy numbers and estimating tumor content ratio (TCR). The HER2/CEP17 ratio is determined by three variables-TCR and absolute copy numbers of HER2 and CEP17-by examining tumor cells; only the ratio of the latter two can be obtained by digital PCR using the whole specimen without purifying tumor cells. TCR was determined by semi-automatic image analysis. We developed a Tumor Content chart, which is a plane of rectangular coordinates consisting of HER2/CEP17 digital PCR data and TCR that delineates amplified, non-amplified, and equivocal areas. By applying this method, 44 clinical gastric cancer biopsy samples were classified as amplified (n = 13), non-amplified (n = 25), or equivocal (n = 6). By comparison, 11 samples were positive, 11 were negative, and 22 were equivocally immunohistochemistry. Thus, our novel method reduced the number of equivocal samples from 22 to 6, thereby obviating the need for confirmation by fluorescence or dual-probe in situ hybridization to < 30% of cases. Tumor content chart-assisted digital PCR analysis is also applicable to multiple sites in surgically resected tissues. These results indicate that this analysis is a useful alternative to HER2 immunohistochemistry in gastric cancers that can serve as a basis for the automated evaluation of HER2 status.

Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms.

Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.

A model for transport phenomena in a cross-flow ultrafiltration module with microchannels.

Cross-flow ultrafiltration of macromolecular solutions in a module with microchannels is expected to have the advantages of fast diffusion from the membrane surface and a high ratio of membrane surface area to feed liquid volume. Cross-flow ultrafiltration modules with microchannels are expected to be used for separation and refining and as membrane reactors in microchemical processes. Though these modules can be applied as a separator connected with a micro-channel reactor or a membrane reactor, there have been few papers on their performance. The purpose of this study was to clarify the relationship between operational conditions and performance of cross-flow ultrafiltration devices with microchannels. In this study, Poly Vinyl Pyrrolidone (PVP) aqueous solution was used as a model solute of macromolecules such as enzymes. Cross-flow ultrafiltration experiments were carried out under constant pressure conditions, varying other operational conditions. The permeate flux decreased in the beginning of each experiment. After enough time passed, the permeate flux reached a constant value. The performance of the module was discussed based on the constant values of the flux. It was observed that the permeate flux increased with increasing transmembrane pressure (TMP) and feed flow rate, and decreased with an increase of feed liquid concentration. A model of the transport phenomena in the feed liquid side channel and the permeation through the membrane was developed based on the concentration and velocity distributions in the feed side channel. The experimental results were compared with those based on the model and the performance of the ultrafiltration module is discussed.