Akihiko Yoshizawa

Somatic mutations affect key pathways in lung adenocarcinoma

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers—including NF1, APC, RB1 and ATM—and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

Characterizing the cancer genome in lung adenocarcinoma

Somatic alterations in cellular DNA underlie almost all human cancers. The prospect of targeted therapies and the development of high-resolution, genome-wide approaches are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumours (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in ∼12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.

Impact of proposed IASLC/ATS/ERS classification of lung adenocarcinoma: prognostic subgroups and implications for further revision of staging based on analysis of 514 stage I cases.

A new lung adenocarcinoma classification is being proposed by the International Association for the Study of Lung Cancer, American Thoracic Society and European Respiratory Society (IASLC/ATS/ERS). This proposal has not yet been tested in clinical datasets to determine whether it defines prognostically significant subgroups of lung adenocarcinoma. In all, 514 patients who had pathological stage I adenocarcinoma of the lung classified according to the Union for International Cancer Control/American Joint Committee on Cancer 7th Edition, and who had undergone a lobectomy with mediastinal lymph node dissection were retrospectively reviewed. Comprehensive histological subtyping was used to estimate the percentage of each histological subtype and to identify the predominant subtype. Tumors were classified according to the proposed new IASLC/ATS/ERS adenocarcinoma classification. Statistical analyses were made including Kaplan–Meier and Cox regression analyses. There were 323 females (63%) and 191 males (37%) with a median age of 69 years (33–89 years) and 298 stage IA and 216 stage IB patients. Three overall prognostic groups were identified: low grade: adenocarcinoma in situ (n=1) and minimally invasive adenocarcinoma (n=8) had 100% 5-year disease-free survival; intermediate grade: non-mucinous lepidic predominant (n=29), papillary predominant (n=143) and acinar predominant (n=232) with 90, 83 and 84% 5-year disease-free survival, respectively; and high grade: invasive mucinous adenocarcinoma (n=13), colloid predominant (n=9), solid predominant (n=67) and micropapillary predominant (n=12), with 75, 7170 and 67%, 5-year disease-free survival, respectively (P<0.001). Among the clinicopathological factors, stage 1B versus 1A (P<0.001), male sex (P<0.008), high histological grade (P<0.001), vascular invasion (P=0.002) and necrosis (P<0.001) were poorer prognostic factors on univariate analysis. Both gross tumor size (P=0.04) and invasive tumor size adjusted by the percentage of lepidic growth (P<0.001) were significantly associated with disease-free survival with a slightly stronger association for the latter. Multivariate analysis showed the prognostic groups of the IASLC/ATS/ERS histological classification (P=0.038), male gender (P=0.007), tumor invasive size (P=0.026) and necrosis (P=0.002) were significant poor prognostic factors. In summary, the proposed IASLC/ATS/ERS classification of lung adenocarcinoma identifies histological categories with prognostic differences that may be helpful in identifying candidates for adjunctive therapy. The slightly stronger association with survival for invasive size versus gross size raises the need for further studies to determine whether this adjustment in measuring tumor size could impact TNM staging for small adenocarcinomas.

Validation of the IASLC/ATS/ERS Lung Adenocarcinoma Classification for Prognosis and Association with EGFR and KRAS Gene Mutations: Analysis of 440 Japanese Patients

Introduction: This study aimed to validate the utility of the new histological classification proposed by the International Association for the Study of Lung Cancer (IASLC), American Thoracic Society (ATS), and European Respiratory Society (ERS) for identifying the prognostic subtypes of adenocarcinomas in Japanese patients; correlations between the classification and the presence of EGFR or KRAS mutation status were also investigated. Methods: We retrospectively reviewed 440 patients with lung adenocarcinoma, who underwent resection. The tumors were classified according to the IASLC/ATS/ERS classification. EGFR and KRAS mutations were detected using the established methods. Results: Five-year disease-free survival rates were: 100% for adenocarcinoma in situ (n = 20) and minimally invasive adenocarcinoma (n = 33), 93.8% for lepidic-predominant adenocarcinoma (n = 36), 88.8% for invasive mucinous adenocarcinoma (n = 10), 66.7% for papillary-predominant adenocarcinoma (n = 179), 69.7% for acinar-predominant adenocarcinoma (n = 61), 43.3% for solid-predominant adencoarcinoma (n = 78), and 0% for micropapillary-predominant adenocarcinoma (n = 19). Multivariate analysis revealed that the new classification was an independent predictor of disease-free survival. EGFR and KRAS mutations were detected in 90 cases (53.9%) and 21 cases (13.3%), respectively; EGFR mutations were significantly associated with adenocarcinoma in situ, minimally invasive adenocarcinoma, lepidic- and papillary-predominant adenocarcinoma, and KRAS mutations adenocarcinomas with mucinous tumor subtypes. Conclusions: We found that the IASLC/ATS/ERS classification identified prognostic histologic subtypes of lung adenocarcinomas among Japanese patients. Histologic subtyping and molecular testing for EGFR and KRAS mutations can help predict patient prognosis and select those who require adjuvant chemotherapy.

A grading system of lung adenocarcinomas based on histologic pattern is predictive of disease recurrence in stage I tumors.

Currently no objective grading system for pulmonary adenocarcinomas exists. To determine whether specific histologic patterns or combinations thereof could be linked to an objective grading system, the histologic patterns in metastatic tumor deposits was compared with the patterns seen in the corresponding 73 primary tumor to determine whether a specific pattern had higher propensity to metastasize. The concordance of the predominant histologic pattern in the primary tumor and the metastases was of 100% for micropapillary, 86% for solid, 42% for acinar, and 23% for papillary types of adenocarcinoma. Informed by these results, a 3-tier grading system based on the histologic subtypes was established. Grade I, a pattern with low metastatic potential (BAC); Grade II, patterns with intermediate metastatic potential (acinar and papillary); and Grade III, patterns with high metastatic potential (solid and micropapillary). These grades were combined into a number of different scoring systems, whose ability to predict recurrence or death from disease was tested in 366 stage 1 adenocarcinomas. A score based on the 2 most predominant grades was able to stratify patients into low-to-high risk for recurrence or death of disease (P=0.001). The 5-years disease-free survival for patients in the highest score group was of 0.73, compared with 0.84 and 0.92 in the intermediate and lowest score groups. Concordance probability estimate was 0.65 (95% confidence interval 0.57-0.73). Therefore, this scoring system provides valuable information in discriminating patients with different risk of disease-recurrence in a highly homogeneous population of patients with stage I cancer.

A grading system combining architectural features and mitotic count predicts recurrence in stage I lung adenocarcinoma

The International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) has recently proposed a new lung adenocarcinoma classification. We investigated whether nuclear features can stratify prognostic subsets. Slides of 485 stage I lung adenocarcinoma patients were reviewed. We evaluated nuclear diameter, nuclear atypia, nuclear/cytoplasmic ratio, chromatin pattern, prominence of nucleoli, intranuclear inclusions, mitotic count/10 high-power fields (HPFs) or 2.4 mm2, and atypical mitoses. Tumors were classified into histologic subtypes according to the IASLC/ATS/ERS classification and grouped by architectural grade into low (adenocarcinoma in situ, minimally invasive adenocarcinoma, or lepidic predominant), intermediate (papillary or acinar), and high (micropapillary or solid). Log-rank tests and Cox regression models evaluated the ability of clinicopathologic factors to predict recurrence-free probability. In univariate analyses, nuclear diameter (P=0.007), nuclear atypia (P=0.006), mitotic count (P<0.001), and atypical mitoses (P<0.001) were significant predictors of recurrence. The recurrence-free probability of patients with high mitotic count (≥5/10 HPF: n=175) was the lowest (5-year recurrence-free probability=73%), followed by intermediate (2–4/10 HPF: n=106, 80%), and low (0–1/10 HPF: n=204, 91%, P<0.001). Combined architectural/mitotic grading system stratified patient outcomes (P<0.001): low grade (low architectural grade with any mitotic count and intermediate architectural grade with low mitotic count: n=201, 5-year recurrence-free probability=92%), intermediate grade (intermediate architectural grade with intermediate–high mitotic counts: n=206, 78%), and high grade (high architectural grade with any mitotic count: n=78, 68%). The advantage of adding mitotic count to architectural grade is in stratifying patients with intermediate architectural grade into two prognostically distinct categories (P=0.001). After adjusting for clinicopathologic factors including sex, stage, pleural/lymphovascular invasion, and necrosis, mitotic count was not an independent predictor of recurrence (P=0.178). However, patients with the high architectural/mitotic grade remained at significantly increased risk of recurrence (high vs low: P=0.005) after adjusting for clinical factors. We proposed this combined architectural/mitotic grade for lung adenocarcinoma as a practical method that can be applied in routine practice.

Overexpression of Phospho-eIF4E Is Associated with Survival through AKT Pathway in Non–Small Cell Lung Cancer

Purpose: The eukaryotic translation initiation factor complex 4E (eIF4E) is downstream in the mammalian target of rapamycin (mTOR) pathway. This study explored expression of eIF4E and its relationship with the PTEN/AKT and RAS/MEK/ERK pathways in non–small cell lung carcinoma (NSCLC). Experimental Design: The status of phosphorylated eIF4E (p-eIF4E), phosphorylated AKT (p-AKT), PTEN, phosphorylated tuberin (p-TSC2), phosphorylated mTOR (p-mTOR), phosphorylated S6 (p-S6), and phosphorylated Erk1/2 (p-Erk1/2) was studied using immunohistochemical analysis applied to a tissue microarray containing 300 NSCLCs. Staining results for each antibody were compared with clinical and pathologic features, and the relationship between staining results was explored. Results: Overexpression of p-eIF4E, p-AKT, p-TSC2, p-mTOR, p-S6, and p-Erk1/2 in NSCLC was found in 39.9%, 78.8%, 5.1%, 46.7%, 27.1%, and 16.6% of tumors, respectively. The phenotype of p-eIF4E correlated positively with that of p-AKT, p-TSC2, and p-S6 (P < 0.001). Overall survival in NSCLC patients was significantly shorter in cases with overexpression of p-eIF4E and p-AKT alone and in combination (log-rank P < 0.001, each). Cases with underexpression of PTEN were limited (6.4%), and this phenotype did not correlate with any clinical variable. In cluster analysis, the p-AKT/p-mTOR/p-eIF4E/p-S6–positive group had significantly shorter survival compared with the survival of all cases (P < 0.001). Multivariate analysis showed that p-eIF4E overexpression is an independent prognostic factor for NSCLC (P = 0.004). Conclusions: This study shows that p-eIF4E expression in addition to p-AKT predicts poor prognosis in NSCLC. Moreover, the correlation between expression of p-eIF4E with p-AKT, as well as p-TSC2 and p-S6, indicates that eIF4E activation through the AKT pathway plays an important role in the progression of NSCLC. Clin Cancer Res; 16(1); OF1–9

Prognostic Significance of Adenocarcinoma in situ, Minimally Invasive Adenocarcinoma, and Nonmucinous Lepidic Predominant Invasive Adenocarcinoma of the Lung in Patients with Stage I Disease

According to the IASLC/ATS/ERS classification, the lepidic predominant pattern consists of 3 subtypes: adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and nonmucinous lepidic predominant invasive adenocarcinoma. We reviewed tumor slides from 1038 patients with stage I lung adenocarcinoma, recording the percentage of each histologic pattern and measuring the invasive tumor size. Tumors were classified according to the IASLC/ATS/ERS classification: 2 were AIS, 34 MIA, and 103 lepidic predominant invasive. Cumulative incidence of recurrence (CIR) was used to estimate the probability of recurrence. Patients with AIS and MIA experienced no recurrences. Patients with lepidic predominant invasive tumors had a lower risk for recurrence (5-y CIR, 8%) than nonlepidic predominant tumors (n=899; 19%; P=0.003). Patients with >50% lepidic pattern tumors experienced no recurrences (n=84), those with >10% to 50% lepidic pattern tumors had an intermediate risk for recurrence (n=344; 5-y CIR, 12%), and those with ⩽10% lepidic pattern tumors had the highest risk (n=610; 22%; P<0.001). CIR was lower for patients with ⩽2 cm tumors than for those with >2 to 3 cm tumors (for both total and invasive tumor size), with the difference more pronounced for invasive tumor size (5-y CIR, 13% vs. 21% [total size; P=0.022] and 12% vs. 27% [invasive size; P<0.001]). Most patients with lepidic predominant adenocarcinoma who experienced a recurrence had potential risk factors, including sublobar resection with close margins (⩽0.5 cm; n=2), 20% to 30% micropapillary component (n=2), and lymphatic or vascular invasion (n=2). It therefore may be possible to identify lepidic predominant adenocarcinomas that carry a low or high risk for recurrence.

Lung involvement in IgG4-related lymphoplasmacytic vasculitis and interstitial fibrosis: report of 3 cases and review of the literature.

There have been a few reports on lung diseases associated with an increased number of infiltrating IgG4-positive plasma cells or lung involvement of IgG4-related sclerosing disease, although their characteristic histologic features have not been well described. Herein, we present 3 cases of interstitial lung disease with common histology and abundant IgG4-positive cell infiltration. Patient 1, a 65-year-old man, was incidentally noted to have a nodular lesion in the left hilar region. In patient 2, a 78-year-old man with dyspnea on effort, computed tomography revealed multifocal consolidation adjacent to the pleura in both lower lobes. Patient 3, a 74-year-old man, underwent lung biopsy for right-sided pleural effusion of unknown etiology. The histologic findings of the 3 individuals were characterized by expansion of the interstitium by dense lymphoplasmacytic infiltrate with prominent vascular involvement, which was indistinguishable from grade 1 lesion of lymphomatoid granulomatosis (LYG-G1). In situ hybridization for Epstein-Barr virus-encoded small RNA (EBER) revealed few or no EBER-positive lymphocytes. Vascular involvement showed subendothelial lymphoplasmacytic infiltrate, including numerous IgG4-positive plasma cells. The percentages of IgG4-positive to IgG-positive plasma cells were 85%, 47%, 46%, respectively. Similar vascular lesions have been described in previous reports of lung complications in IgG4-related sclerosing diseases. Some of these lesions were diagnosed as LYG-G1 without EBER-positive cells. In conclusion, LYG-G1–like lesion characterized by lymphoplasmacytic vasculitis might be a characteristic feature of IgG4-related lung disease.

Predictors of Outcomes after Surgical Treatment of Synchronous Primary Lung Cancers

Introduction: Distinguishing synchronous primary lung cancers (SPLCs) from advanced disease is important because prognosis and treatments are very different and a surgical approach to SPLC may result in survival similar to solitary cancers. Determining this distinction with certainty, however, is challenging. We reviewed our experience with surgical resection of presumed SPLC to analyze outcomes and identify factors associated with prolonged survival. Patients and Methods: A retrospective review identified patients treated for presumptive SPLC. Cases were defined using modified criteria set forth by Martini and Melamed and histologic subtyping. Survival was estimated using the Kaplan-Meier method, and factors associated with survival were evaluated using a log-rank test or Cox proportional hazards model for categorical and continuous variables, respectively. Results: From January 1995 to July 2006, 175 patients met study criteria and underwent complete resection. Tumors were more often in different lobes of an ipsilateral chest (55 of 175, 31%) or contralateral lesions (45 of 175, 26%). More than half (104 of 175, 59%) of the patients underwent a single operation. Median follow-up was 50.3 months (4.8-164.7); median overall survival (OS) for the group was 67.4 months (46.4-80.0) with a 3-year OS of 64%. On multivariable analysis controlling for stage, only female gender was a significant predictor of better OS (p = 0.001). Conclusions: An aggressive surgical approach to patients with apparent SPLC can result in survival that is comparable with patients with single lung cancers of similar stage. The Martini and Melamed criteria and histologic subtyping can identify appropriate patients for resection. Female gender was associated with superior OS.