Prognostic value of tumor-infiltrating lymphocytes differs depending on histological type and smoking habit in completely resected non-small-cell lung cancer.

BACKGROUND: T-cell infiltration in tumors has been used as a prognostic tool in non-small-cell lung cancer (NSCLC). However, the influence of smoking habit and histological type on tumor-infiltrating lymphocytes (TILs) in NSCLC remains unclear. PATIENTS AND METHODS: We evaluated the prognostic significance of TILs (CD4+, CD8+, CD20+, and FOXP3+) according to histological type and smoking habit using automatic immunohistochemical staining and cell counting in 218 patients with NSCLC. RESULTS: In multivariate survival analyses of clinical, pathological, and immunological factors, a high ratio of FOXP3+ to CD4+ T cells (FOXP3/CD4) [hazard ratio (HR): 4.46, P < 0.01 for overall survival (OS); HR: 1.96, P < 0.05 for recurrence-free survival (RFS)] and a low accumulation of CD20+ B cells (HR: 2.45, P = 0.09 for OS; HR: 2.86, P < 0.01 for RFS) were identified as worse prognostic factors in patients with adenocarcinoma (AD). In non-AD, a low number of CD8+ T cells were correlated with an unfavorable outcome (HR: 7.69, P < 0.01 for OS; HR: 3.57, P < 0.02 for RFS). Regarding smoking habit in AD, a high FOXP3/CD4 ratio was poorly prognostic with a smoking history (HR: 5.21, P < 0.01 for OS; HR: 2.38, P < 0.03 for RFS), whereas a low accumulation of CD20+ B cells (HR: 4.54, P = 0.03 for OS; HR: 2.94, P < 0.01 for RFS) was confirmed as an unfavorable factor in non-smokers with AD. CONCLUSIONS: A low number of CD8+ T cells in non-AD, a high FOXP3/CD4 ratio in smokers with AD, and a low number of CD20+ B cells in non-smokers with AD were identified as independent unfavorable prognostic factors in resected NSCLC. Evaluating the influence of histological type and smoking habit on the immunological environment may lead to the establishment of immunological diagnosis and appropriate individualized immunotherapy for NSCLC.

RET-rearranged non-small-cell lung carcinoma: a clinicopathological and molecular analysis.

BACKGROUND: To elucidate clinicopathological characteristics of non-small-cell lung carcinoma (NSCLC) cases carrying RET rearrangements causing oncogenic fusions to identify responders to therapy with RET tyrosine kinase inhibitors. METHODS: We investigated 1874 patients with carcinomas, including 1620 adenocarcinomas (ADCs), 203 squamous cell carcinomas (SCCs), 8 large cell carcinomas, and 43 sarcomatoid carcinomas (SACs). Fluorescence in situ hybridisation (FISH) and/or reverse transcription-PCR (RT-PCR) were performed to detect RET gene rearrangement. RESULTS: In all, 22 cases (1.2%) showed RET rearrangements; all cases were of ADC histology. Of the 22 patients, 19 possessed KIF5B-RET fusion genes, whereas 3 possessed CCDC6-RET fusion genes. The RET-rearranged tumours were significantly more common in younger patients (P=0.038) and tended to occur in patients with no history of smoking (P=0.051). In addition, RET rearrangements were not associated with gender, occupational history (particularly radioactive exposure), tumour size, lymph node status, tumour stage, or patient survival. The predominant growth pattern in RET-rearranged ADCs was lepidic in 6 cases, papillary in 9 cases, acinar in 2 cases, micropapillary in 1 case, and solid in 4 cases. Cells with cytoplasmic mucin production were at least focally present in 12 of the 22 (54.5%) RET-rearranged ADC cases. Among the 21 analysed RET-rearranged tumours, RET immunopositivity was observed in 15 cases (71.4%), and was significantly associated with RET rearrangement (P<0.001). CONCLUSIONS: The RET rearrangements were observed in 1.2% of NSCLCs. All cases of RET rearrangement were ADCs. The RET rearrangements were more likely to be observed in younger patients. Although cytoplasmic mucin production was at least focally present in 54.5% of RET-rearranged ADCs, specific histological features were not detected.

Clinicopathological features of nonsmall cell lung carcinomas with BRAF mutations.

BACKGROUND: Recently, driver tyrosine kinase gene mutations have been detected in malignant tumors, including lung tumors. Notwithstanding their attractiveness as targets for molecular therapy, limited information is available regarding BRAF-mutated lung carcinomas. MATERIALS AND METHODS: BRAF mutation status was determined in 2001 surgically resected nonsmall-cell lung cancer (NSCLC) cases using high-resolution melting analysis (HRMA) followed by Sanger sequencing and/or deep sequencing using next generation sequencer. RESULTS: BRAF mutations were detected in 26 (1.3%) of 2001 NSCLC cases (25 adenocarcinomas and 1 squamous cell carcinoma). In the 26 cases, 13 mutation genotypes were identified, including V600E (8 of 26; 30.8%), G469A (6 of 26; 23.1%), K601E (4 of 26; 15.4%), and other residual mutations (1 of 26; 0.04%). Of the 13 genotypes, 4 genotypes (G464E, G596R, A598T, and G606R) had not been previously reported in lung cancer. The overall survival rate was not significantly different between patients with wild-type BRAF and those with V600E or non-V600E BRAF mutations (P = 0.49 and P = 0.15, respectively). Histomorphological analysis revealed that focal clear cell changes were present in 75% of V600E-mutated tumors. All V600E BRAF-mutated tumors were negative for other driver gene alterations including epidermal growth factor receptor (EGFR) and KRAS mutations and the anaplastic lymphoma kinase gene translocation, whereas five tumors with non-V600E BRAF mutations (four G469A and one G464E/G466R) showed concomitant EGFR mutations. CONCLUSION: The frequency of BRAF mutations in lung cancer was low in an Asian cohort. Furthermore, BRAF mutation status lacked prognostic significance in this patient population.

Distinct outcome of stage I lung adenocarcinoma with ACTN4 cell motility gene amplification.

BACKGROUND: Even if detected at an early stage, a substantial number of lung cancers relapse after curative surgery. However, no method for distinguishing such tumors has yet been established. PATIENTS AND METHODS: The copy number of the actinin-4 (ACTN4) gene was determined by fluorescence in situ hybridization on tissue microarrays comprising 543 surgically resected adenocarcinomas of the lung. RESULTS: Amplification (an increase in the copy number by ≥ 2.0 fold) of the ACTN4 gene was detected in two of seven lung adenocarcinoma cell lines and 79 (15%) of 543 cases of pathological stage I-IV lung adenocarcinoma. Multivariate analysis revealed that ACTN4 gene amplification was the most significant independent factor associated with an extremely high risk of death (hazard ratio, 6.78; P = 9.48 × 10(-5), Cox regression analysis) among 290 patients with stage I lung adenocarcinoma. The prognostic significance of ACTN gene amplification was further validated in three independent cohorts totaling 1033 patients. CONCLUSIONS: Amplification of the ACTN4 gene defines a small but substantial subset of patients with stage I lung adenocarcinoma showing a distinct outcome. Such patients require intensive medical attention and might benefit from postoperative adjuvant chemotherapy.

Diagnostic and prognostic significance of the alternatively spliced ACTN4 variant in high-grade neuroendocrine pulmonary tumours.

BACKGROUND: High-grade neuroendocrine tumours (HGNTs) of the lung manifest a wide spectrum of clinical behaviour, but no method for predicting their outcome has been established. MATERIALS AND METHODS: We newly established a monoclonal antibody specifically recognizing the product of the alternatively spliced ACTN4 transcript (namely, variant actinin-4), and used it to examine the expression of variant actinin-4 immunohistochemically in a total of 609 surgical specimens of various histological subtypes of lung cancer. RESULTS: Variant actinin-4 was expressed in 55% (96/176) of HGNTs, but in only 0.8% (3/378) of non-neuroendocrine (NE) lung cancers. The expression of variant actinin-4 was significantly associated with poorer overall survival in HGNT patients (P=0.00021, log-rank test). Multivariate analysis using the Cox proportional hazards model showed that the expression of variant actinin-4 was the most significant independent negative predictor of survival in HGNT patients (hazard ratio (HR), 2.15; P=0.00113) after the presence of lymph node metastasis (HR, 2.25; P=0.00023). CONCLUSIONS: The expression of variant actinin-4 is an independent prognostic factor for patients with HGNTs. This protein has a high affinity for filamentous actin polymers and likely promotes aggressive behaviour of cancer cells. The present clinical findings clearly support this notion.

Protein expression and gene copy number changes of receptor tyrosine kinase in thymomas and thymic carcinomas.

BACKGROUND: Insulin-like growth factor-1 receptor (IGF-1R), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-type 2 (HER2), and c-Met are members of the receptor tyrosine kinases (RTKs). The associations between the RTK status [protein expression and gene copy number (GCN)] and patient characteristics and between the RTK status and prognosis remain undetermined. MATERIALS AND METHODS: The study included 140 patients who underwent surgery for thymic tumors. Protein expression was evaluated by immunohistochemistry (IHC) and GCN was evaluated by bright-field in situ hybridization (BISH). The correlations between the RTK status and clinicopathological findings were examined. RESULTS: IGF-1R protein was frequently detected in thymic carcinoma (83.8%) and EGFR in thymic tumors (91.4%). Thirty-six and 39 tumors were BISH high for IGF-1R and EGFR, respectively: 28 and 25 exhibited high polysomy; 8 and 14 exhibited gene amplification. No tumor was positive for HER2 or c-Met by IHC and BISH. Multivariate analysis revealed that IGF-1R gene amplification (P = 0.027), thymic carcinoma histology, and higher tumor stage were significantly correlated with an adverse prognosis. CONCLUSIONS: Thymic epithelial tumors frequently express IGF-1R and/or EGFR proteins. IGF-1R gene amplification is suggested to define an unfavorable subset for thymic epithelial tumors.

Surgical implications of the new IASLC/ATS/ERS adenocarcinoma classification.

A new adenocarcinoma classification was recently introduced by a joint working group of the International Association for the Study of Lung Cancer (IASLC), American Thoracic Society (ATS) and European Respiratory Society (ERS). A distinction is made between pre-invasive lesions, and minimally invasive and invasive adenocarcinoma. The confusing term "bronchioloalveolar carcinoma" is not used any more and new subcategories include adenocarcinoma in situ and minimally invasive adenocarcinoma. Due to a renewed interest in screen-detected nodules and early-stage lung cancers of <2 cm, this classification also has profound implications for thoracic surgeons. In this article, surgical topics are discussed: the role of a minimally invasive approach, especially video-assisted thoracic surgery, limited resection for early-stage lung cancer, the extent of lymph node dissection, the accuracy of intraoperative frozen section analysis, management of multiple lung nodules and prognostic factors in operated patients. Specific key issues are presented based on the current evidence and areas of surgical uncertainty are defined providing a basis for further studies. Thoracic surgeons will play a major role in the application and global introduction of this new adenocarcinoma classification. The remaining controversies regarding the precise diagnosis and management of early-stage lesions will have to be resolved by multidisciplinary and international collaboration.

DEK oncoprotein regulates transcriptional modifiers and sustains tumor initiation activity in high-grade neuroendocrine carcinoma of the lung.

Lung cancer shows diverse histological subtypes. Large-cell neuroendocrine cell carcinoma and small-cell lung carcinoma show similar histological features and clinical behaviors, and can be classified as high-grade neuroendocrine carcinoma (HGNEC) of the lung. Here we elucidated the molecular classification of pulmonary endocrine tumors by copy-number profiling. We compared alterations of copy number with the clinical outcome of HGNEC and identified a chromosomal gain of the DEK oncogene locus (6p22.3) that was significantly associated with poor prognosis. We further confirmed that DEK overexpression was associated with poor prognosis in a larger set of HGNEC. Downregulation of DEK by small hairpin RNA led to a marked reduction of in vitro colony formation, in vivo tumorigenicity and chemo-resistance, and was associated with loss of lung cancer stem cell markers. Gene expression profiling revealed that DEK downregulation was associated with altered expression of transcriptional regulators, which specifically include known targets of interchromosomal translocations in hematopoietic tumors, and knockdown of these epigenetic modifiers affected colony formation activity. Our study showed that DEK overexpression, partly through an increase in its gene dose, mediates the activity of global transcriptional regulators and is associated with tumor initiation activity and poor prognosis in HGNEC.

Population data on 10 non-CODIS STR loci in Japanese population using a newly developed multiplex PCR system.

This paper describes a newly devised autosomal short tandem repeat (STR) multiplex polymerase chain reaction (PCR) systems for 10 loci (D1S1656, D2S1353, D8S1132, D12S1090, D14S608, D18S535, D19S253, D20S480, D21S226, and D22S689) unlinked to the core STR loci (non-CODIS loci). Of 252 samples taken from the Japanese population, PCR products ranged in length from 107 bp to 319 bp. No significant deviations from Hardy-Weinberg equilibrium were observed at any of the 10 loci. The accumulated power of discrimination and power of exclusion for the 10 loci were 0.999999999998 and 0.99991, respectively. We conclude that the present multiplex system for the 10 non-CODIS loci represents a powerful tool for forensic applications.

A randomised phase II trial of preoperative chemotherapy of cisplatin-docetaxel or docetaxel alone for clinical stage IB/II non-small-cell lung cancer results of a Japan Clinical Oncology Group trial (JCOG 0204).

Preoperative chemotherapy is a promising strategy in patients with early-stage resectable non-small-cell lung cancer (NSCLC); optimal chemotherapy remains unclear. Clinical (c-) stage IB/II NSCLC patients were randomised to receive either two cycles of docetaxel (D)-cisplatin (P) combination chemotherapy (D 60 mg m(-2) and P 80 mg m(-2) on day 1) every 3-4 weeks or three cycles of D monotherapy (70 mg m(-2)) every 3weeks. Thoracotomy was performed 4-5 weeks (DP) or 3-4 weeks (D) after chemotherapy. The primary end point was 1-year disease-free survival (DFS). From October 2002 to November 2003, 80 patients were randomised. Chemotherapy toxicities were mainly haematologic and well tolerated. There were two early postoperative deaths with DP (one intraoperative bleeding and one empyema). Pathologic complete response was observed in two DP patients. Docetaxel-cisplatin was superior to D in terms of response rate (45 vs 15%) and complete resection rate (95 vs 87%). Both DFS and overall survival were better in DP. Disease-free survival at 1, 2 and 4 years were 78, 65 and 57% with DP, and were 62, 44 and 36% with D, respectively. Preoperative DP was associated with encouraging resection rate and DFS data, and phase III trials for c-stage IB/II NSCLC are warranted.

Evaluation of miniY-STR multiplex PCR systems for extended 16 Y-STR loci.

We developed three short amplicon Y-chromosomal short tandem repeat (miniY-STR) polymerase chain reaction multiplex systems for 16 Y-STR loci (DYS441, DYS446, DYS462, DYS481, DYS485, DYS495, DYS505, DYS510, DYS511, DYS549, DYS 575, DYS578, DYS593, DYS618, DYS638, and DYS643), using newly designed primer sets. In an assay of 238 Japanese males using the three miniY-STR systems, amplification product lengths ranged from 91 to 151 bp for all 16 Y-STR loci. We identified 212 different haplotypes among the 238 individuals, finding haplotype diversity and discrimination capacity of 0.9974 and 0.8908, respectively. An assay of degraded DNA samples using the three miniY-STR multiplex systems, including artificially degraded samples and degraded forensic casework samples, proved remarkably effective. In conclusion, analyses of miniY-STR multiplex systems will play an important role in forensic applications involving degraded DNA samples for which genotyping using only commercial kits is ill-suited.

MiniSTR multiplex systems based on non-CODIS loci for analysis of degraded DNA samples.

We describe two short amplicon autosomal short tandem repeat (miniSTR) quadruplex systems for eight loci D1S1171, D2S1242, D3S1545, D4S2366, D12S391, D16S3253, D20S161, and D21S1437, unlinked from the combined DNA index system (non-CODIS) loci, using newly designed primer sets. The results of an assay of 411 Japanese individuals showed that polymerase chain reaction (PCR) products within the eight loci were less than 150bp in size, without the seven additional bases for adenylation. The frequency distributions in the loci showed no deviations from Hardy-Weinberg equilibrium expectations. The accumulated power of discrimination and power of exclusion for the eight loci were 0.9999999991 and 0.998, respectively. For assay of highly degraded DNA, including artificially degraded samples and the degraded forensic casework samples assessed with the present miniSTR quadruplex systems, the systems proved quite effective in analyzing degraded DNA.

Solitary pulmonary granuloma caused by Mycobacterium avium-intracellulare complex.

OBJECTIVES: To analyse the clinical features and high resolution computed tomography (HRCT) findings of solitary pulmonary granulomas caused by the Mycobacterium avium-intracellulare (MAI) complex. METHODS: We retrospectively analysed a series of 73 consecutive patients with solitary pulmonary granuloma and negative sputum smear and culture results, in whom the diagnosis was established by histological examination of specimens obtained by partial pulmonary resection or lobectomy. We compared the clinical features and HRCT findings of the solitary pulmonary granulomas definitively diagnosed to be caused by the MAI complex with those of granulomas of other causes by univariate and multivariate analyses. RESULTS: In this study series of 24 patients with solitary pulmonary granuloma, the aetiological agent was established as being the MAI complex. According to the results of the multivariate analysis, 'female sex', 'pleural indentation' and 'lobulation' on the HRCT images were significantly associated with solitary pulmonary granuloma caused by the MAI complex. CONCLUSION: This study demonstrated several characteristics of solitary pulmonary granulomas caused by the MAI complex, and suggested that it might be a subtype of pulmonary MAI complex infection without the typical radiographic features of the infection.

MiniY-STR quadruplex systems with short amplicon lengths for analysis of degraded DNA samples.

Two short amplicon Y-chromosomal short tandem repeat (miniY-STR) quadruplex systems for the eight Y-STR loci DYS522, DYS508, DYS632, DYS556, DYS570, DYS576, DYS504 and DYS540 were devised using newly designed primer sets. Among 224 samples from Japanese population, amplification product lengths detected in these Y-STR loci ranged from 95 to 147bp, while 170 different haplotype were identified (discrimination capacity=0.7589 and haplotype diversity=0.9949). As a result of test on degraded DNA samples using the miniY-STR quadruplex systems, the systems proved to be an quite effective tools for analyzing degraded DNAs. We conclude that analyses of the miniY-STR quadruplex systems in addition to the commercial available Y-STR multiplex kits are highly useful for forensic practices of degraded DNA samples.

Diagnostic accuracy of CT-guided percutaneous cutting needle biopsy for thymic tumours.

AIM: To determine the diagnostic accuracy of computed tomography (CT)-guided percutaneous cutting needle biopsy (PCNB) for thymic tumours in accordance with the World Health Organization (WHO) classification. MATERIAL AND METHODS: We retrospectively analysed a consecutive series of 138 cases in which CT-guided PCNB had been performed for an anterior mediastinal tumour. Its sensitivity and specificity for thymic epithelial tumours were evaluated, and the concordance between the histopathological diagnosis according to the WHO classification of thymic tumours based on PCNB and the diagnosis is based on the surgical specimens was assessed by Kappa statistic. RESULTS: The diagnostic sensitivity and specificity of CT-guided PCNB for thymic tumours were 93.3 and 100%, respectively. The overall concordance between the diagnosis according to the WHO classification established by PCNB specimen and by the surgical specimen was 79.4% (weighted kappa=0.79). CONCLUSION: CT-guided PCNB is a reliable method of diagnosing thymic tumours, and there was good concordance for the WHO classification between the diagnosis based on CT-guided PCNB specimen and that based on the surgical specimen.

Japanese population data for eight X-STR loci using two new quadruplex systems.

Using two new X-chromosome short tandem repeats (X-STR) multiplex (quadruplex) systems, we investigated Japanese population data (drawn from 401 individuals) for eight X-STR loci (DXS10011, DXS9898, DXS8377, HPRTB, DXS7132, DXS6797, GATA172D05, and DXS6807). Allele typing with the systems was successful for all loci. The combined powers of discrimination of the eight loci in men and women were 0.999997 and 0.9999999996, respectively. We conclude that combined analyses of the eight X-STR loci using these two quadruplex polymerase chain reaction systems represent a powerful tool for Japanese forensic practice.

MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis.

We sought to evaluate a more effective system for analyzing X-chromosomal short tandem repeats (X-STRs) in highly degraded DNA. To generate smaller amplicon lengths, we designed new polymerase chain reaction (PCR) primers for DXS7423, DXS6789, DXS101, GATA31E08, DXS8378, DXS7133, DXS7424, and GATA165B12 at X-linked short tandem repeat (STR) loci, devising two miniX-multiplex PCR systems. Among 333 Japanese individuals, these X-linked loci were detected in amplification products ranging in length from 76 to 169 bp, and statistical analyses of the eight loci indicated a high usefulness for the Japanese forensic practice. Results of tests on highly degraded DNA indicated the miniX-STR multiplex strategies to be an effective system for analyzing degraded DNA. We conclude that analysis by the current miniX-STR multiplex systems offers high effectiveness for personal identification from degraded DNA samples.

Allele frequencies of the six miniSTR loci in a population from Japan.

Allele frequencies and forensic parameters for the six miniSTR loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434, and D22S1045 were investigated in a sample of 142 unrelated healthy Japanese individuals. The polymerase chain reaction (PCR) products contained within the six loci were less than 119 bp in size. The frequency distributions in the six short tandem repeat (STR) loci showed no deviations from Hardy-Weinberg equilibrium expectations. The accumulated powers of discrimination and power of exclusion for the six loci were 0.999998 and 0.98, respectively. It was thus considered that due to the small PCR products and the moderate degree of polymorphism, analysis with use of the six miniSTR loci was highly beneficial for the forensic analysis of degraded DNA.

Distant failure after treatment of postoperative locoregional recurrence of non-small cell lung cancer.

BACKGROUND: The standard treatment for patients with locoregional recurrence of non-small cell lung cancer (NSCLC) after complete resection has not been established. The aim of this study was to evaluate clinicopathologic characteristics, type of locoregional recurrence, pattern of subsequent failure, and survival after the recurrence. METHODS: Of 743 patients undergoing pulmonary resection for NSCLC in the National Cancer Center Hospital between 1990 and 1995, we retrospectively reviewed the medical charts of the 43 patients (5.8 %) found to have locoregional recurrence without distant metastasis or pleural or pericardial involvement. RESULTS: The median time to locoregional recurrence was 13.6 months (range: 1.6 - 85.8 months). The most frequent site of recurrence was the mediastinal nodes in 21 of 43 patients (49 %). 33 patients (77 %) received further treatment for the recurrence: thoracic irradiation in 26, surgery in two, systemic chemotherapy in two, and a combination of the above in 3 patients. Subsequent distant failure was detected in 26 (68 %) of the 38 patients assessable for the analysis of failure pattern: lung in 11, brain in 6, bone in 5, and others in 13. The median interval from the recurrence to distant failure was 8.4 months (range: 1.7-56.4 months). The median survival time after diagnosis of the locoregional recurrence was 10.5 months (range: 0-74.0 months). A multivariate analysis showed that local therapy for the locoregional recurrence had no significant impact on postrecurrent survival or distant failure-free survival. CONCLUSIONS: Many patients with postoperative locoregional recurrence developed distant metastases early after the first recurrence. Systemic chemotherapy in addition to local therapy may be of benefit in this population.