Yuko Minami

Hepatocyte Growth Factor Expression in EGFR Mutant Lung Cancer with Intrinsic and Acquired Resistance to Tyrosine Kinase Inhibitors in a Japanese Cohort

Introduction: This study was performed to determine the incidence rates of resistance factors, i.e., high-level hepatocyte growth factor (HGF) expression, epidermal growth factor receptor (EGFR) T790M secondary mutation, and MET amplification, in tumors with intrinsic and acquired EGFR tyrosine kinase inhibitor (TKI) resistance in EGFR mutant lung cancer. Methods: Ninety-seven specimens from 93 EGFR mutant lung cancer patients (23 tumors with acquired resistance from 20 patients, 45 tumors with intrinsic resistance from 44 patients [nonresponders], 29 sensitive tumors from 29 patients) from 11 institutes in Japan were analyzed. HGF expression, EGFR T790M secondary mutation, and MET amplification were determined by immunohistochemistry, cycleave real-time polymerase chain reaction, and fluorescence in situ hybridization, respectively. Results: High-level HGF expression, EGFR T790M secondary mutation, and MET amplification were detected in 61, 52, and 9% of tumors with acquired resistance, respectively. High-level HGF expression was detected in 29% of tumors with intrinsic resistance (nonresponders), whereas EGFR T790M secondary mutation and MET amplification were detected in 0 and 4%, respectively. HGF expression was significantly higher in tumors with acquired resistance than in sensitive tumors (p < 0.001, Students t test). Fifty percent of tumors with acquired resistance showed simultaneous HGF expression with EGFR T790M secondary mutation and MET amplification. Conclusions: High-level HGF expression was detected more frequently than EGFR T790M secondary mutation or MET amplification in tumors with intrinsic and acquired EGFR-TKI resistance in EGFR mutant lung cancer in Japanese patients. These observations provide a rationale for targeting HGF in EGFR-TKI resistance in EGFR mutant lung cancer.

Reproducibility of histopathological subtypes and invasion in pulmonary adenocarcinoma. An international interobserver study

Histological subtyping of pulmonary adenocarcinoma has recently been updated based on predominant pattern, but data on reproducibility are required for validation. This study first assesses reproducibility in subtyping adenocarcinomas and then assesses further the distinction between invasive and non-invasive (wholly lepidic) pattern of adenocarcinoma, among an international group of pulmonary pathologists. Two ring studies were performed using a micro-photographic image-based method, evaluating selected images of lung adenocarcinoma histologic patterns. In the first study, 26 pathologists reviewed representative images of typical and ‘difficult’ histologic patterns. A total number of scores for the typical patterns combined (n=94) and the difficult cases (n=21) were 2444 and 546, respectively. The mean kappa score (±s.d.) for the five typical patterns combined and for difficult cases were 0.77±0.07 and 0.38±0.14, respectively. Although 70% of the observers identified 12–65% of typical images as single pattern, highest for solid and least for micropapillary, recognizing the predominant pattern was achieved in 92–100%, of the images except for micropapillary pattern (62%). For the second study on invasion, identified as a key problem area from the first study, 28 pathologists submitted and reviewed 64 images representing typical as well as ‘difficult’ examples. The kappa for typical and difficult cases was 0.55±0.06 and 0.08±0.02, respectively, with consistent subdivision by the same pathologists into invasive and non-invasive categories, due to differing interpretation of terminology defining invasion. In pulmonary adenocarcinomas with classic morphology, which comprise the majority of cases, there is good reproducibility in identifying a predominant pattern and fair reproducibility distinguishing invasive from in-situ (wholly lepidic) patterns. However, more precise definitions and better education on interpretation of existing terminology are required to improve recognition of purely in-situ disease, this being an area of increasing importance.

Non-small-cell lung cancer and Ba/F3 transformed cells harboring the ERBB2 G776insV_G/C mutation are sensitive to the dual-specific epidermal growth factor receptor and ERBB2 inhibitor HKI-272.

Mutation-specific cancer therapy has shown promising clinical efficacy. In non-small-cell lung cancer (NSCLC), the presence of mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase correlates with clinical response to small-molecule tyrosine kinase inhibitors. Here, we show that cells harboring the G776insV_G/C mutation in the related ERBB2 tyrosine kinase (also known as HER2 or Neu), present in a small percentage of NSCLCs, are sensitive to HKI-272, an irreversible dual-specific kinase inhibitor targeting both EGFR and ERBB2. In the ERBB2-mutant NCI-H1781 cell line, HKI-272 treatment inhibited proliferation by induction of G(1) arrest and apoptotic cell death. Furthermore, HKI-272 abrogated autophosphorylation of both ERBB2 and EGFR. Finally, Ba/F3 murine pro-B cells, engineered to express mutant ERBB2, became independent of interleukin-3 and sensitive to HKI-272. Thus, the subset of NSCLC patients with tumors carrying the ERBB2 G776insV_G/C mutation may benefit from treatment with HKI-272.

Allele-dependent variation in the relative cellular potency of distinct EGFR inhibitors

Targeted cancer therapies impede cancer cell growth by inhibiting the function of activated oncogene products. Patients with non-small cell lung cancer and somatic mutations of EGFR can have a dramatic response to treatment with erlotinib and gefitinib; different somatic mutations are associated with different times to progression and survival. In this study, the relative and absolute potencies of two distinct EGFR tyrosine kinase inhibitors, erlotinib and an investigational irreversible inhibitor, HKI-272, were found to vary significantly in a panel of Ba/F3 cells transformed by representative EGFR somatic mutations. HKI-272 more potently inhibited the primary exon 20 insertion mutants, the secondary erlotinib-resistance mutants including T790M and many erlotinib-sensitive mutants including L858R. In contrast, erlotinib is a more potent inhibitor of the major exon 19 deletion mutants than is HKI-272. Analyses of EGFR autophosphorylation patterns confirmed the mutation-specific variation in relative potency of these tyrosine kinase inhibitors. Our finding that distinct EGFR inhibitors are more effective in vitro for different mutant forms of the protein suggests that tyrosine kinase inhibitor treatment could be tailored to specific EGFR mutations. More broadly, these results imply that the development and deployment of targeted therapies should focus on inhibition of specific cancer-causing mutations, not only on the mutated target.

Bronchioloalveolar carcinoma (lepidic growth) component is a more useful prognostic factor than lymph node metastasis.

Introduction: Although many factors predictive of patient survival have been reported for lung cancer, no comparative studies have attempted to determine those that are most significant for practical medicine. Methods: We conducted a retrospective review of 139 patients who underwent complete resection of adenocarcinomas less than 2 cm in diameter between 1993 and 2000 at the National Cancer Center Hospital (Tokyo, Japan). The MIB-1 labeling index (LI), immunohistochemical staining for carcinoembryonic antigen (CEA), p53, p27, epidermal growth factor receptor (EGFR), phosphorylated-EGFR (pEGFR), Cox-2, neuronatin, &ggr;H2AX, and thyroid transcription factor-1 (TTF-1), the prevalence of a micropapillary pattern, and the ratio of the bronchioloalveolar cell carcinoma (BAC) or lepidic growth (LG) component were determined, and their significance as prognostic factors for lung adenocarcinoma was compared. Results: Univariate analysis showed that lymph node metastasis (p-N status), BAC/LG component, vascular invasion (p-V status), MIB-1 LI, pEGFR, and CEA were prognostically significant (p-N status: p < 0.0001, BAC/LG: p = 0.0005, p-V status: 0.002, MIB-1 LI: p = 0.005, pEGFR: p = 0.024, and CEA: p = 0.049). Multivariate analysis showed that only p-N status (p = 0.013) was of prognostic significance. However, BAC/LG component (p = 0.051) was a more reliable prognostic factor than p-N status in mixed adenocarcinoma with a BAC/LG component. Conclusion: In comparison with other immunohistochemical and histopathologic factors, BAC/LG component is independently and reliably prognostic for small adenocarcinoma of the lung, and, in particular, for the major histologic subtype (adenocarcinoma mixed subtype with BAC/LG), BAC/LG component is more reliably prognostic than lymph node metastasis.

Nuclear Grading of Primary Pulmonary Adenocarcinomas: Correlation Between Nuclear Size and Prognosis

According to the World Health Organization Classification of Tumors, the prognostic value of morphometric cytologic atypia has not been assessed in pulmonary adenocarcinoma.

Expression of HNFs and C/EBPα is correlated with immunocytochemical differentiation of cell lines derived from human hepatocellular carcinomas, hepatoblastomas and immortalized hepatocytes

Objective assessment of the differentiation grade of hepatocellular carcinomas (HCCs) is important for evaluation of the pathological diagnosis, prognosis and therapeutic treatment. Differentiation of hepatocytes is reflected by their expression of hepatic functional proteins in the mouse embryo, and liver‐enriched transcription factors (LETFs) have been shown to regulate hepatic functional genes strictly. Previous reports demonstrated that the level of LETF expression is altered in HCC or preneoplastic nodules compared with noncancerous tissues. Therefore, LETF expression levels might be useful as a measure of HCC maturation. In this study, to clarify the correlation between the expression of LETFs and the differentiation grade of HCCs, we performed a quantitative analysis of the mRNA expressions of HNFs and C/EBPα using real‐time reverse‐transcription PCR and immunocytochemical analysis for hepatic functional proteins in twelve cell lines. Furthermore, we examined orthotopic transplantations of the HCC cell lines in C.B‐17/Icrj‐scid/scid mice and characterized the histologic and cytologic differentiation of the tumors that developed. Our results showed that comprehensive expressions of HNF‐3β, HNF‐4α, HNF‐1α, and C/EBPα were specific to HCCs with well‐differentiated function and morphology. Furthermore, among these four transcription factors, HNF‐4α and HNF‐1α expressions showed synchronism and had a close relation with HCC differentiation. These in vitro results were confirmed in tumors developed in SCID mice in vivo. These findings suggested that HNF‐4α and HNF‐1α are useful markers to assess the degree of HCC differentiation, which we suggest could be evaluated objectively by the quantitative analysis of HNFs and C/EBPα in HCCs.

High expression of stratifin is a universal abnormality during the course of malignant progression of early‐stage lung adenocarcinoma

Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis, with a 5‐year survival rate of 100%. However, early invasive adenocarcinoma (EIA) often has a fatal outcome. In this study, we compared the expression profiles of AIS with those of EIA showing lymph node metastasis or a fatal outcome, and screened the differentially expressed genes by cDNA microarray. From the genes selected, we focused on Stratifin (SFN, 14‐3‐3 σ), which showed significantly higher expression in EIA than in AIS. Immunohistochemistry for SFN revealed that more than 95% of EIAs were immunopositive for SFN, in comparison to only 13% of AISs (p <0.05). Interestingly, positivity was detected not only in the invasive region but also in the in situ spreading component of EIA. Functionally, SFN facilitates the cell proliferation capacity of lung adenocarcinoma. These results indicate that SFN overexpression is a universal abnormality during the stepwise progression from in situ to invasive adenocarcinoma of the lung.

Overexpression of immunoglobulin (CD79a) binding protein1 (IGBP-1) in small lung adenocarcinomas and its clinicopathological significance

Immunoglobulin binding protein 1 (IGBP‐1) was initially identified as a signal transduction molecule coprecipitating with MB1 (Igα) of the B cell antigen receptor (BCR) complex and was later found to be broadly expressed. Immunoglobulin binding protein 1 has been characterized as an associated and regulatory component of the catalytic subunits of protein phosphatase 2A (PP2A), which is the most abundant phosphatase and plays important roles in cell growth and cell cycle control. The aim of this study was to investigate the expressional characteristics of IGBP‐1 and PP2Ac during pulmonary adenocarcinogenesis. The positivity rate of IGBP‐1 increased during the course of sequential progression from non‐invasive carcinoma (8/30, 26.7%) to invasive adenocarcinoma (37/46, 80.4%) among cases that showed areas of lepidic growth. In contrast, all of the small adenocarcinomas showing a non‐lepidic growth pattern were positive for IGBP‐1 (20/20, 100%). All cancers that proved ultimately fatal were positive for IGBP‐1, and log‐rank analysis showed that IGBP‐1 positivity was significantly correlated with a poor prognosis. In contrast, atypical adenomatous hyperplasias and lung adenocarcinomas were uniformly positive for PP2Ac. Protein phosphatase 2A was not associated with carcinoma progression. Thus we have demonstrated that IGBP‐1 is expressed universally in advanced lung adenocarcinomas, and that its overexpression is significantly related to outcome.

Reproducibility of the diagnosis of small adenocarcinoma of the lung and usefulness of an educational program for the diagnostic criteria.

Using 32 small adenocarcinomas of the lung including bronchioloalveolar carcinoma (BAC), the reproducibility of diagnosis by the modified diagnostic criteria for small adenocarcinoma (Cancer 75; 2844, 1995) and the effectiveness of an educational program for 27 volunteer general pathologists were examined. The average coincidence rate of the diagnosis before and after the program was 42.4% and 56.6%, respectively. The coincidence rate after the program was significantly higher than that before the program (P < 0.05). In contrast, the average coincidence rate of six lung cancer specialists was 71.4%, and this was significantly higher than that for general pathologists after the program (P < 0.05). When the cases were divided into two groups (in situ adenocarcinoma (BAC and BAC with alveolar collapse) and early invasive adenocarcinoma), the average coincidence rate for the general pathologists after the program increased to 85.3%, which was significantly higher than that before the program (80.3%; P < 0.05). The rate for the specialists was 89%, which was higher than that for the general pathologists after the program but not significantly so. This trial was thought to provide a theoretical background for the histological diagnosis of peripheral type adenocarcinoma of the lung and to justify the existing diagnostic criteria.