Hideki Takakura

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.

Rapid Detection of Epidermal Growth Factor Receptor Mutations in Lung Cancer by the SMart-Amplification Process

Purpose: A positive response to gefitinib in non–small cell lung cancer (NSCLC) has been correlated to mutations in epidermal growth factor receptor (EGFR) gene. Previous reports have been based mainly on diagnostic screening by sequencing. However, sequencing is a time-consuming and complicated procedure, not suitable for routine clinical use. Experimental Design: We have developed rapid, simple, and sensitive mutation detection assays based on the SMart Amplification Process (SMAP) and applied it for analyzing EGFR gene mutations in clinical samples. By using SMAP, we can detect mutations within 30 min including sample preparation. To validate the assay system for potential use in clinical diagnostics, we examined 45 NSCLC patients for EGFR mutations using sequencing and SMAP. Results: The outcomes of the SMAP assay perfectly matched the sequencing results, except in one case where SMAP was able to identify a mutation that was not detected by sequencing. We also evaluated the sensitivity and specificity of SMAP in mutation detection for EGFR. In a serial dilution study, SMAP was able to find a mutation in a sample containing only 0.1% of the mutant allele in a mixture of wild-type genomic DNA. We also could show amplification of mutated DNA with only 30 copies per reaction. Conclusions: The SMAP method offers higher sensitivity and specificity than alternative technologies, while eliminating the need for sequencing to identify mutations in the EGFR gene of NSCLC. It provides a robust and point-of-care accessible approach for a rapid identification of most patients likely to respond to gefitinib.

Importance of Complete Pathologic Response to Prehepatectomy Chemotherapy in Treating Colorectal Cancer Metastases

Objective:We studied the influence of complete pathologic response of colorectal cancer liver metastases to prehepatectomy chemotherapy on long-term survival after hepatectomy. Summary Background Data:Although complete response seen on imaging may be a useful criterion for evaluating efficacy of chemotherapy, complete clinical response by imaging has shown limited predictive value for complete pathologic response in treating colorectal liver metastases. Methods:We retrospectively analyzed data from 63 patients who received preoperative chemotherapy and underwent hepatectomy. Results:Of 472 liver metastases evaluated, 86 were no more visible from images after chemotherapy. We excluded 14 of these metastasis treated with local ablation. Of the remaining 72 metastasis, 22 (30.6%) were microscopically persistent metastases or recurrences in situ. Liver metastases with complete pathologic response had smaller diameters at diagnosis than others (P < 0.001), and microscopic cancer deposits surrounding macroscopic tumors were less frequent in patients with complete pathologic response than others (P < 0.05). Outcomes were favorable in patients whose liver metastases all showed a complete pathologic response. Even patients with complete pathologic response in only some metastases showed higher overall and disease-free survival rates than pathologic nonresponders (P = 0.001 and P = 0.002, respectively). Presence or absence of metastases showing complete pathologic response was an independent prognostic factor (relative risk, 4.464; P = 0.0099). Conclusions:Little correlation was observed between imaging response of colorectal cancer liver metastases to chemotherapy and pathologic response. Liver surgery should be undertaken even after a complete response by imaging. Outcome after hepatectomy was favorable in patients showing complete pathologic response of least one metastasis.

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.

Technique of right hemihepatectomy preserving ventral right anterior section guided by area of hepatic venous drainage

BACKGROUND Although the consequences of partial venous outflow interruption have attracted only limited attention in liver surgery, maximal preservation of liver function after hepatic resection requires preservation of circulation in the remnant liver, especially hepatic vein drainage. METHODS Data from 30 patients undergoing 3-dimensional imaging were analyzed to clarify the relationship between the area of the ventral right anterior section (RAS) and that drained by regional hepatic vein tributaries. The feasibility of our preliminary technique of right hemihepatectomy preserving the ventral RAS also was evaluated. RESULTS The median estimated volume of the ventral RAS was 230 mL (range, 88-391). The average ratio of this estimated volume of the ventral RAS to total estimated liver volume was 18.0 +/- 4.9%. The median volume of the territory served by middle hepatic vein (MHV) tributaries draining the ventral RAS, expressed as a percentage of the whole volume of the ventral RAS, was 82.5%. Findings in fusion images of portal and hepatic vein territories demonstrated an area of MHV tributaries comparable with the ventral RAS area in 73.3% of all cases. As for the results of right hemihepatectomy with the ventral RAS preserved, no tumor was exposed on transection surfaces, and no recurrence took place within the preserved ventral RAS of the remnant liver. CONCLUSION Procedures considering the importance of regional venous drainage offer the possibility of reducing the extent of surgery without loss of effectiveness.

Small hepatocellular carcinoma with ring calcification: a case report and literature review.

Ring calcification in untreated hepatocellular carcinoma (HCC) is extremely rare, with only 3 previously reported cases in the English-language literature. A case of HCC with ring calcification was reported in this paper. Additionally, 3 previously reported cases of HCC with ring calcification were reviewed. In 3 of these 4 cases (including our case), surgery was performed. Although the size of the ring-calcified lesion ranged from 3.0-3.7 cm in previously reported cases, the size was only 1 cm in ours. The differentiation of the tumor was moderate in the 2 previously reported cases in the histological findings and poor in ours. In spite of their poor differentiation for their sizes, these tumors showed no early enhancement in dynamic computed tomography. All calcified tumors showed a thick fibrous capsule and extensive necrosis histologically. Ring calcification was considered to result from a circulatory disturbance caused by the imbalance between the less abundant arterial blood flow and high inner pressure induced by either the thick fibrous capsule or vigorous proliferation due to the poor differentiation. Ring calcification in untreated HCC may suggest a lower differentiation of the tumor. Even if its size is small, hepatic resection should be performed for any tumor with ring calcification because poor differentiation is considered to be one of the risk factors for recurrence after local ablation therapy, including radio frequency ablation.

Utilization of Distilled Water Lavage for Localized Fluid Collection After Combined Hepatectomy and Cyst Fenestration for Polycystic Liver Disease

Ascites necessitating persistent drainage or paracentesis after drain removal has been found among early postoperative complications after combined hepatectomy and cyst fenestration (CHCF) for polycystic liver disease (PLD). It has been reportedly observed in 20% to 70% of cases and seemed to easily cause recurrent symptoms unless properly treated. We utilized distilled water lavage for treating localized fluid collection after combined hepatectomy and cyst fenestration for PLD. A 63-year-old female patient underwent CHCF for PLD, which caused severe abdominal fullness. Early postoperative course was uneventful until 10 days after surgery when the patient suffered sudden abdominal fullness and resultant severe anorexia because of right subphrenic massive localized monolocular fluid collection diagnosed by abdominal computed tomography—although total liver volume was reduced to less than half of that before surgery. Percutaneous drainage relieved symptoms immediately, but the drainage tube could not be rem...