[Detection of ALK positive pulmonary adenocarcinoma using immunostaining].

Although ALK-positive lung cancer cases have been recently reported, it is impossible to detect using only morphology technique. Furthermore, though RT-PCR and FISH techniques can be used for detection, they are not practical for screening. We investigated whether ALK-positive lung cancer could be detected using a conventional immunostaining method. Resected lung adenocarcinoma samples from 88 nonsmoker cases were selected and screening was performed using ALK immunostaining in 24 cases that did not have the EGFR or k-ras mutation. We found that the optimal staining condition was treatment using a water bath and detection with a Novo Link Polymer Detection System (Leica microsystems). Of the 24 cases examined, ALK expression was found in 4, of which ALK separated signals were found in 3 using a FISH method. No separated signals were seen in cases with negative immunostaining findings. Detection by immunostaining was found useful for ALK mutated lung cancer cases, though the pretreatment and detection methods utilized are important.

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.

Clinicopathologic roles of tumor-infiltrating lymphocytes and CD8-positive lymphocytes in lung cancer imprint smears in squamous cell carcinoma and adenocarcinoma.

OBJECTIVE: To investigate the presence of tumor-infiltrating lymphocytes (TILs) and CD8-positive lymphocytes (CD8s) in lung cancers and to examine the prognostic significance of their relationship with clinicopathologic parameters. STUDY DESIGN: Primary tumor imprint smears from 83 lung cancers were consecutively obtained at surgery at Tsuchiura Kyodo General Hospital between 1996 and 1998. TILs were observed in Papanicolaou-stained smears, and CD8s were immunocytochemically visualized. RESULTS: TILs were judged positive in 42 cases (51%), and 34 of 83 cases (41%) were positive for CD8s. There was no correlation between the frequency of TILs and CD8s. TILs assessed by Papanicolaou staining in 83 cases showed no correlation with any clinicopathologic factors. CD8s were observed more frequently in squamous cell carcinomas (SCCs) than other adenocarcinomas. A high frequency of CD8s was associated with lymph node metastasis (p = 0.043), stage (p = 0.025) and tumor differentiation (p = 0.03). TILs and CD8s made no significant difference in survival. However, in SCC the patients positive for TILs but negative for CD8s survived and had a significantly better prognosis than did the others (p = 0.037). CONCLUSION: CD8s were associated with tumor progression and development of malignancy in lung cancers. Especially in SCC, the cases had a high frequency of TILs. A low number of CD8s may correlate with a favorable prognosis.

Immunohistochemical detection of telomerase reverse transcriptase (TERT) protein in benign and malignant tumor cells of the breast.

Telomerase activity is associated with immortality and is expressed in various malignant tumors. Telomerase reverse transcriptase (TERT) is a human telomerase catalytic subunit and is strongly correlated with telomerase activity. In this study, the expression of TERT was investigated by immunocytochemistry to determine whether it is a useful marker for differential diagnosis in benign or malignant breast tumors. We examined imprint smears prepared from 63 surgically resected breast lesions, and 27 cases of secretion or aspiration cytology smears had been routinely diagnosed. Eighteen of the 63 cases were examined for TERT mRNA and the correlation with expression of TERT protein was investigated. Expression of TERT protein was correlated with expression of TERT mRNA by in situ hybridization (p=0.044), suggesting that telomerase activity might be expressed with morphologic factors. In imprint smears and routine cytology smears, TERT-positive cases significantly tended to be malignant cases (p=0.014, p=0.033 respectively). Especially, in malignant imprint smear cases, positive expression of TERT was significantly correlated with lymph node metastasis (p=0.022). In conclusion, the expression of TERT protein might be a useful marker for differential diagnosis in malignant and benign breast tumors. In malignant cases, it could be a useful indicator for the assessment of potential of lymph node metastasis [corrected].

The usefulness of immunostaining in washed cell cytological smears for differential diagnosis of B cell type malignant lymphoma.

We investigated the usefulness of immunostaining on washed cell cytological smears for the differential diagnosis of B-cell type malignant lymphoma. Twenty-eight cases with possible malignant lymphoma were examined. The tissues were squashed in a test tube of isotonic saline. The cell suspensions were then washed to remove non-specific soluble immmunoglobulin, and were smeared. Immunocytochemical staining for IgG, IgA, IgM, kappa and lambda was performed on the smears. As seen in the results, all cases of B cell lymphoma have a positive rate of 20% or over for kappa or lambda, and a kappa/lambda ratio of over 4.5 or a ratio of over 2.0 lambda/kappa, in the washed cell smears. Thus, it is possible to distinguish B cell lymphoma from the others by means of the results use as cut off scores. In addition, the rapid staining protocol in the present study was able to provide the results within 15 min. Therefore, it might be the most suitable method for the detection of immunoglobulin monoclonality in general laboratories.

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.

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.

Blood flow does not correlate with the size of metastasis in our new intravital observation model of Lewis lung cancer.

We previously reported a novel in situ observation model for microcirculation of lung metastasis from subcutaneously implanted Lewis lung cancer into mouse. Using this model, we studied the correlation of blood flow and the size of lung metastasis. It was revealed that metastatic growth and its angiogenesis are suppressed by circulating angiogenesis inhibitors, such as angiostatin or endostatin, released from primary tumor. When we removed the primary tumor, the metastasized lung cancer significantly grew faster and larger. But the blood flow per area did not increase either inside or outside of the metastatic tumor. This suggests that the growth of metastatic tumor is directly regulated not by blood flow increase but by the other effects of the circulating factors.