Naoya Funakoshi

Preservation of pancreatic beta cell function with pulsatile cardiopulmonary bypass

Pancreatic islet cell function and tissue metabolism were studied during and after cardiopulmonary bypass in 38 patients undergoing an open heart operation. Twenty patients were operated on with pulsatile cardiopulmonary bypass (group I) and 18, with nonpulsatile cardiopulmonary bypass (group II). Hyperglycemia was observed during and early after operation in both groups. In group I during cardiopulmonary bypass, the immunoreactive insulin and c-peptide levels and the insulin to glucagon molar ratio increased significantly compared with the preoperative values, but in group II, these variables did not alter significantly. An hour postoperatively, the immunoreactive insulin (71 +/- 34 muIU/mL) and c-peptide (8.3 +/- 3.0 ng/mL) levels and the insulin to glucagon molar ratio (11.0 +/- 5.2) in group I were significantly higher than those in group II (immunoreactive insulin, 29 +/- 20 muIU/mL; c-peptide, 4.8 +/- 1.8 ng/mL; insulin to glucagon molar ratio, 3.4 +/- 2.6). The blood lactate level in group I (41 +/- 22 mg/dL) was significantly lower than that in group II (78 +/- 30 mg/dL) an hour postoperatively. In conclusion, pulsatile cardiopulmonary bypass is quite effective in preserving pancreatic beta cell function and tissue metabolism during and early after open heart procedures.

Detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification

For pathological diagnoses, visualization of genetic status using routine tissue sections is important to determine the relationships between histopathological findings and genetic alterations. Loop‐mediated isothermal amplification (LAMP) has been reported to have high levels of specificity and amplification efficiency. An in situ LAMP method was used, along with an amplification refractory mutation system (ARMS) to directly detect a specific point mutation, L858R, which is a mutation of epidermal growth factor receptor (EGFR), useful for the prediction of the effects of the anti‐lung cancer drug gefitinib. The investigation was done using two types of cultured cells as well as paraffin‐sectioned specimens collected from 26 cases of surgically resected lung cancer. Twelve of the specimens had an L858R mutation and in situ LAMP showed reactions in the nuclei of all cancer cells present in those. Such reactions were also shown on in situ LAMP in three of the remaining 14 cases that were without the L858R mutation. In addition, a few cases showed responses in the nuclei of bronchial epithelium cells located in non‐cancerous areas in the vicinity of a positive tumor, which suggested that the mutation had already occurred in the tumorigenic early stage. It is concluded that the present method is useful for pathological and genetic diagnoses.

Correlation between EGFR gene mutation pattern and Akt phosphorylation in pulmonary adenocarcinomas

The detection of gene mutation of epithelial growth factor receptor (EGFR) is important to predict the therapeutic effect of gefitinib. Recently, it was reported that examination of the activation of the downstream protein of EGFR is useful in the same way as the EGFR mutation. Therefore the purpose of the present paper was to determine whether activation of Akt and Erk, which are downstream proteins, and the EGFR gene mutation pattern was correlated. A total of 130 pulmonary adenocarcinomas were studied for the gene mutations of EGFR in exon 19 and 21, and the phosphorylation of Akt and Erk was investigated by immunostaining. The EGFR mutation was detected in 32%, the positivity of p‐Akt was 51%, and the rate of p‐Erk was 27%. The EGFR mutation‐positive cases were the minority in p‐Akt‐negative cases, and the p‐Akt expression was significantly associated with the mutation of EGFR (P = 0.0014). In addition, there was a significant correlation between the L858R mutation and the expression of p‐Akt (P = 0.040). It is suggested that the activation of Akt is dependent on EGFR mutation pattern.

Prognostic significance of gastric cancer metastasis in second-tier lymph nodes detected on reverse transcriptase–polymerase chain reaction and immunohistochemistry

To determine the prognostic significance of the methods used to determine the presence of metastasis in second‐tier lymph nodes of patients with gastric cancer, the authors studied lymph nodes surgically removed from 100 patients with gastric cancer (55 with early cancer, 45 with progressive). The results of HE staining were compared with those of immunohistochemistry using the anticytokeratin (CK) antibody and reverse transcriptase–polymerase chain reaction (RT‐PCR) assays. Lymph node 7 or 8a was obtained intraoperatively, then mRNA was extracted using an immunobeads method, and RT‐PCR with CK19 mRNA was performed. The P for Cox regression analysis for metastasis detected by HE staining, CK staining, and RT‐PCR of all 100 cases was 0.312, 0.426, and 0.021, respectively, while for second‐tier lymph nodes it was 0.154, 0.013, and 0.006, respectively. In conclusion, RT‐PCR and CK staining for detection of metastasis in second‐tier lymph nodes were more reliable prognostic indicators than conventional HE staining.

Usefulness of epidermal growth factor receptor and p53 cocktail immunostaining for differential diagnosis with urine cytology.

OBJECTIVE To investigate immunochemical markers of assisting cytologic diagnosis to evade the inaccuracy to be caused by alteration of urinary samples. STUDY DESIGN We used p53 and epidermal growth factor receptor (EGFR) in an immunostaining cocktail for the differential diagnosis of urine cytology. We reviewed urothelial tumor tissues resected from 36 cases, normal urothelium tissues collected from 20 autopsy cases and 108 separate urine samples. RESULTS No positive case was obtained from the autopsy cases. In 20 of the cancer cases treated by transurethral resection, the average positive rates of EGFR and p53 cells were 69% and 12%, respectively. As for the urine smears, positive findings were observed in atypical cells in 20 of 21 cytologically positive cases (95%), which were also confirmed to be malignant in histologic examinations. In contrast, color development was observed in urothelial cells from only 9 of 87 cases (10%), which were unclear for atypia and judged benign, though 5 of those 9 specimens were from patients with a previous history of urothelial cancer. In 2 of those 5 cases, recurrences were confirmed later. CONCLUSION For urine cytology, we found that our cocktail immunostaining method was useful for the differential diagnosis.

Usefulness of broncoalveolar lavage samples for detection of Pneumocystis carinii using PCR method

We use PCR to diagnose Pneumocystis carinii (PC) pneumonia together with cytology. In this report, we studied the difference in detection of PC between PCR and cytology, sputum, and broncoalveolar lavage samples suspicious for PC pneumonia. First, we made cytology smears from samples and treated them with Papanicolaou and Grocott stainng. DNA extractation from remnant samples and amplification of DNA specific for PC were also done. Using PCR, we detected PC in 12 of 22 (55%) samples of broncoalveolar lavage, but only 4 of 33 (12%) sputum samples (p=0.0018). In cytology, we similarly detected 6 cases (27%), but only 1 case (3%)(p=0.013). Broncoalveolar lavage samples were thus more suitable than sputum in detecting PC. PCR was significantly more sensitive than cytology (p=0.039). Grocott staining is generally used and is important for detecting PC. However, it could not confirm positive in case of insufficiency of PC on the smears. We concluded that detection using broncoalveolar lavage samples using PCR were suitable for detecting PC.

The expression of MMP-7 in colon cancer and its clinical application.

To study the clinical significance of MMP-7 in colorectal cancers, we investigated 69 cases of imprint smear using immunocytochemistry and in situ hybridization. A total of 39 of 69 cases were positive for MMP-7 expression by immunocytochemistry. The expression of MMP-7 protein and mRNA were correlated. Positive cases of MMP-7 proteintented to be moderately differentiated tumors of the sigmoid colon or rectum, and invasive cases. The expression of MMP-7 and metastases to lymph nodes and liver were strongly correlated. In conclusion, MMP-7 may be a useful marker for invasion and metastasis in colorectal cancer.

Re-evaluation of the Classification of Atypical Cells and the Method of Post-screening Management in Sputum Cytology Mass Screening.

喀痰検診5年間の成績を踏まえて, より効率的な早期肺扁平上皮癌の発見をめざした異型扁平上皮細胞判定基準と事後指導の方法を検討することを目的とした.延べ37015名の受診者のうち, 肺癌学会分類を基にして作成した筆者らの細胞判定基準による判定C (中等度異型) からの癌の発見はなかったが, 判定D (高度異型扁平上皮および癌疑い) では, 高度異型扁平上皮の精検受診者77名から4名 (5.2%), 癌疑いでは36名から10名 (27.8%) の癌が発見された. 従って, 判定Cでは次年度の喀痰検診による追跡で十分であると思われ, 判定Dでは, 癌が疑われると判定された場合には直ちに精検が必要だが, それよりも異型が弱く高度異型と判定された場合には直ちに喀痰細胞診を再検し, 細胞異型が同様に比較的弱ければ, 以後喀痰細胞診と胸部X線写真で追跡すればよいものと思われた.

Cytology of the non-neoplastic pulmonary diseases with a tumorous shadow on a chest roentgenogram.

胸部X線写真上肺野腫瘤状陰影を呈した2例の非腫瘍性肺良性疾患の擦過細胞診に出現した異型細胞について検討した. 第1例は4O歳主婦の器質化肺炎例で, 核小体の著しく肥大した大型の類円形の核を有する異型細胞を認めた. この細胞の核所見からのみでは高分化腺癌との鑑別が困難であったが, 細胞の数が少なく結合性の強いことが悪性との鑑別の要点であると考えられた. 第2例は33歳の主婦で, 肺硬塞発症後3～4日めに核小体の目立つ未熟な扁平上皮化生細胞に類似した細胞を認め, 組織修復細胞との関連が考えられた.