Yuji Takeda

Analysis of expression patterns of breast cancer-specific markers (mammaglobin and gross cystic disease fluid protein 15) in lung and pleural tumors.

CONTEXT The lung is the most common site of metastasis during the natural history of malignant tumors. Breast carcinoma has a propensity for distant metastasis, and the lung and pleura are among the most common metastatic sites. Although it is often difficult to make a clear-cut differential diagnosis between the two, distinguishing primary lung carcinoma from breast carcinoma metastatic to the lung is important because the treatment modalities are different. OBJECTIVE To elucidate the utility of mammaglobin and gross cystic disease fluid protein 15 (GCDFP-15), which are known to be breast-specific antigens, in distinguishing various primary lung and pleural tumors from breast carcinoma metastasizing to the lung. DESIGN A total of 20 cases of breast carcinoma metastatic to the lung and 263 tumors of nonbreast origin located in the lung and pleura were analyzed. RESULTS Of the 20 cases of breast carcinoma metastatic to the lung, 10 (50.0%) were immunoreactive for mammaglobin and 9 (45.0%) for GCDFP-15, the frequency of positivity being slightly higher for the former than for the latter. The area immunopositive for mammaglobin showed more diffuse staining than the area immunopositive for GCDFP-15. Furthermore, the specificity of mammaglobin for breast carcinoma metastatic to the lung was superior (98.9%) to that of GCDFP-15 (91.8%). CONCLUSION The sensitivity of mammaglobin is equal or superior to that of GCDFP-15 for investigation of breast carcinoma. Immunopositivity for mammaglobin is more diffuse than that for GCDFP-15. In terms of practical diagnosis, mammaglobin immunohistochemistry can serve as a differential marker of breast carcinoma and should be added to the immunohistochemical panel.

Co-existence of positive MET FISH status with EGFR mutations signifies poor prognosis in lung adenocarcinoma patients

MET, a receptor tyrosine kinase for hepatocyte growth factor, is associated with tumor progression and acquired resistance to epidermal growth factor tyrosine kinase inhibitors (EGFR-TKI). Therefore, MET gene alterations could be both prognostic and predictive. Fluorescence in situ hybridization (FISH) is one method for assessing gene alteration, but the frequency of positive cases varies due to a lack of standardized criteria. We evaluated MET gene copy number in lung adenocarcinoma and its association with clinicopathological characteristics. FISH was applied to evaluate high MET gene copy number and true amplification in 138 lung adenocarcinoma patients using two criteria: the Cappuzzo scoring system and PathVysion. MET positive cases according to the Cappuzzo scoring system evidenced both aneuploidy and true amplification, whereas PathVysion revealed only amplification. Proportion of MET FISH positive cases was 15% and 4% determined by the Cappuzzo system and PathVysion, respectively. PathVysion demonstrated higher frequencies of MET FISH positives among men and smokers and evidenced no MET FISH positives in patients with bronchioloalveolar carcinoma. Prognosis was significantly associated with MET FISH positive only as defined by the PathVysion system (gene amplification), not by the Cappuzzo system. However, progression-free survival time of patients with both EGFR mutations and MET FISH positive defined by the Cappuzzo scoring system was significantly shorter than with EGFR mutations alone. These results suggest that MET FISH is a potential prognostic factor and coexistence of MET FISH with EGFR mutations signifies worse prognosis.

Comparison of different clones (WT49 versus 6F-H2) of WT-1 antibodies for immunohistochemical diagnosis of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is known to mimic the morphology of a number of diverse neoplastic conditions. WT-1 protein is conventionally used as a positive mesothelioma marker. Recently, a new monoclonal antibody clone WT49 has recently become commercially available. To compare specificity and sensitivity of the conventionally used clone 6F-H2 for the diagnosis of MPM to those of the new clone WT49. Forty cases of MPM, and 55 cases of lung carcinoma, 10 cases of synovial sarcoma of the intrathoracic region were analyzed. Of the 40 cases of MPM tested, clone WT49 and 6F-H2 stained 30 (75.0%) and 26 (65.0%) cases, respectively. Nuclear staining of clone WT49 was observed in 4 (7.2%) cases of lung carcinomas and in 1 (10.0%) case of synovial sarcoma. However, there was no nuclear staining of clone 6F-H2 in lesions other than MPM. There was no cytoplasmic staining of clone WT49 in any tumor. However, cytoplasmic staining of clone 6F-H2 was observed in 7 (17.5%) cases of MPM, 17 (30.1%) cases of lung carcinomas, and 5 (50.0%) cases of synovial sarcoma. The main advantage of WT49 is its higher reactivity with the sarcomatoid area of biphasic mesothelioma, but the results also indicate 1 drawback, that this clone was seen to react with a small percentage of lung carcinomas when it is used to distinguish epithelioid mesotheliomas from lung carcinomas. Furthermore, the positive reaction of clone WT49 was restricted to nucleus without cytoplasmic staining, which is seen in conventionally used WT-1 antibodies.

SPARC is a possible predictive marker for albumin-bound paclitaxel in non-small-cell lung cancer

Objectives Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) produced good tumor response in cases with lung squamous cell carcinoma, one of the most difficult cancers to treat. Secreted protein acidic and rich in cysteine (SPARC) binds to albumin, suggesting that SPARC plays an important role in tumor uptake of nab-paclitaxel. There is as yet no predictive marker for cytotoxic agents against non-small-cell lung cancer (NSCLC), and hence we believed that SPARC expression might be associated with tumor response to nab-paclitaxel. Patients and methods We studied stromal SPARC reactivity and its association with clinicopathological characteristics in 200 cases of NSCLC using a custom tissue microarray fabricated in our laboratory by immunohistochemical staining. We also investigated the relationship between stromal SPARC reactivity and tumor response to nab-paclitaxel using biopsy or surgical specimens obtained from advanced or recurrent lung cancer patients. Results High SPARC stromal reactivity (>50% of optical fields examined) was detected in 16.5% of cases and intermediate SPARC reactivity (10%–50%) in 56% of cases. High expression in cancer cells was rare (five cases). Stromal SPARC level was correlated with smoking index, squamous cell carcinoma, and vessel invasion. Furthermore, patients with high stromal SPARC reactivity in biopsy specimens such as transbronchial lung biopsy or surgical specimens tended to respond better to nab-paclitaxel. Conclusion Stromal SPARC was detected by immunohistochemical staining in ∼70% of NSCLC cases, and good tumor response to nab-paclitaxel was correlated with high stromal SPARC reactivity. SPARC may be a useful predictive marker for selecting patients likely to respond favorably to nab-paclitaxel treatment.

Usefulness of plasma HGF level for monitoring acquired resistance to EGFR tyrosine kinase inhibitors in non-small cell lung cancer.

Monitoring of molecular markers is indispensable for deciding subsequent treatment after acquired resistance to molecular-targeted therapy. According to results using re-biopsy, EGFR T790M mutation and overexpression of hepatocyte growth factor (HGF) are major mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). The aim of the present study was to assess whether quantification of HGF using peripheral blood in addition to detection of T790M with plasma DNA is useful for monitoring as an alternative to invasive re-biopsy. HGF levels in plasma were determined using ELISA and T790M mutation was detected using mutation-biased PCR and quenched probe system (MBP-QP). The median level of HGF in plasma at baseline was 140 pg/ml and was significantly higher in the advanced stage of cancer and in smokers and predicted poor survival as determined using 315 plasma samples from 225 lung cancer patients. T790M was detected with plasma DNA in 9 of 16 patients who acquired resistance to EGFR-TKIs and a greater than 1.5-fold elevation compared with pretreatment HGF levels was observed in 6 patients after acquired resistance. Eleven of 16 patients (69%) showed either HGF elevation or T790M in plasma samples, with both outcomes observed in 25% of patients; this is consistent with results based on re-biopsy reported from other laboratories. Considering these results, assessing HGF and T790M using peripheral blood could be useful for monitoring mechanisms of acquired resistance to EGFR-TKIs.

A fully integrated, automated and rapid detection system for KRAS mutations.

KRAS mutations are detected in tumors of various organs, and they are also markers of resistance for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and monoclonal antibodies against the EGFR. Thus, the accurate and rapid detection of KRAS mutations is crucial, not only for screening, but also for the prediction of the efficacy of molecular-targeted therapy. The aim of the present study was to establish a novel automated detection system for KRAS mutations. One hundred and thirty-six lung adenocarcinoma patients were genotyped for KRAS mutations with both the conventional direct sequence (DS) method and with the newly developed quenching probe (QP) method that obtains data automatically within 60 min. The detection limit of the QP method using a control plasmid containing the KRAS mutation was 50 copies, and 10% mutant plasmid was detected in the mixture of wild-type and mutants. The results obtained by the QP and DS methods were identical in all but two of the 136 cases. The two differentially identified samples, which consisted of substantially fewer lung cancer cells, were positive according to the QP method but negative as determined by DS for KRAS mutations. These findings characterize the QP method as an accurate and rapid detection system for KRAS mutations.

A Case of Primary Pulmonary Colloid Adenocarcinoma: How Can We Obtain a Precise Diagnosis?

A 76-year-old asymptomatic man was found to have a mass in the right lower lung field. Although the presence of a mucinous component in the majority of the tumor was shown by magnetic resonance imaging, the presence of cancer cells was suspected by contrast enhancement on computed tomography (CT) and based on the partial accumulation in the marginal regions of the tumor on fluorodeoxyglucose-positron emission tomography (FDG-PET). A transbronchial lung biopsy was non-diagnostic, but resection of the mass resulted in a diagnosis of colloid adenocarcinoma. The findings from combined contrast CT and FDG-PET may raise the suspicion of colloid adenocarcinoma and prompt the consideration of surgical resection.

Current Status and Problems of T790M Detection, a Molecular Biomarker of Acquired Resistance to EGFR Tyrosine Kinase Inhibitors, with Liquid Biopsy and Re-biopsy

Background/Aim: The purpose of this study was to consider appropriate application of liquid and re-biopsy through analysis of current status in practice. Patients and Methods: We performed a retrospective analysis of 22 patients with epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer who exhibited 1st/2nd generation EGFR-tyrosine kinase inhibitors resistance. The cobas® method was used to detect T790M with re-biopsy and the mutation-biased PCR and quenched probe method was used with liquid biopsy. Results: T790M detection rate was 52% with re-biopsy and 58% with liquid biopsy. The concordance between tissue and plasma was 58%. One patient who was T790M-positive with liquid biopsy showed heterogeneity among metastatic lesions in terms of osimertinib efficacy, as revealed by T790M detection with re-biopsy. Conclusion: Liquid biopsy reflects the whole body, whereas re-biopsy is useful for spatial diagnosis. Considering these characteristics, a combination of liquid and re-biopsy contribute to enhanced treatment.

Abstract 1504: Usefulness of peripheral blood for monitoring of acquired resistance to EGFR tyrosine kinase inhibitors in NSCLC

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Determination of mechanisms of acquired resistance to molecular targeted therapy such as EGFR tyrosine kinase inhibitors (EGFR-TKI) is indispensable for overcoming the resistance to the treatment. According to the results using re-biopsy, EGFR T790M mutation, and overexpression of hepatocyte growth factor (HGF) are major mechanisms of acquired resistance to EGFR-TKI in non-small cell lung cancer. Since the biological characteristics of cancer cells could be altered during treatment, it is necessary to clarify the molecular events in each individual on time point of acquired resistance to EGFR-TKI for selection of the appropriate treatment. Considering that, we chose peripheral blood for monitoring molecular alterations contributing to the resistance to EGFR-TKI such as T790M and HGF overexpression. Since amount of circulating tumor DNA (ctDNA) is small, we have newly developed a fully-automated sensitive method, mutation-biased PCR and quenched probe (MBP-QP) system for detection of T790M. HGF was quantified using ELISA. Using these systems, we retrospectively analyzed T790M and HGF levels in peripheral blood in 36 non-small cell lung cancer patients who were treated with EGFR-TKI. Before treatment of EGFR-TKI, HGF levels in plasma samples were less than lower limit of HGF quantification, 100 pg/ml, in 4 patients, and ranged up to 381 pg/ml. T790M was not detected with ctDNA in any patients. The progression free survival (PFS) time and overall survival (OS) time were compared between two groups, high HGF group (more than median) and low HGF group (median and less than that), was investigated. There were no differences of PFS and OS between two populations, suggesting that HGF level in plasma before treatment with EGFR-TKI was neither a predictive marker for anti-cancer effect of EGFR-TKI nor a prognostic marker among these population. Sixteen sets of plasma before treatment of EGFR-TKI and after acquired resistance were obtained. Plasma HGF levels ranged from 90 to 680, and 79 to 1235 pg/ml before treatment of EGFR-TKI, and after acquired resistance, respectively. The ratio of HGF levels after acquired resistance to before treatment was 0.52 to 7.3, and 6 patients showed elevation of HGF 1.5 times and more than that. T790M was detected with plasma DNA in 9 patients after acquired resistance to EGFR-TKI. Eleven of 16 patients (69%) showed either HGF elevation (1.5-fold≦) or T790M with plasma, and both elevations were observed in 4 patients (25%) , which were equivalent to the results using re-biopsy reported in other laboratories. These results of retrospective analysis suggest that quantification of HGF and detection of T790M using peripheral blood are promising systems for monitoring the mechanisms of acquired resistance to EGFR-TKI. Citation Format: Naomi Kobayashi, Naoko Sueoka-Aragane, Hitomi Umeguchi, Tomomi Nakamura, Akemi Sato, Kazutoshi Komiya, Yuji Takeda, Shinichiro Hayashi, Eisaburo Sueoka, Shinya Kimura. Usefulness of peripheral blood for monitoring of acquired resistance to EGFR tyrosine kinase inhibitors in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1504. doi:10.1158/1538-7445.AM2014-1504