Correlation between genetic alterations and histopathological subtypes in bronchiolo-alveolar carcinoma and atypical adenomatous hyperplasia of the lung

Bronchiolo-alveolar carcinoma (BAC) is a type of lung adenocarcinoma characterized by growth along the alveolar wall. It is divided into two subtypes: sclerosing BAC (SBAC), which has central fibrosis, and non-sclerosing BAC (NSBAC), which lacks central fibrosis. We compared the genetic alterations in these two types of BAC with those in atypical adenomatous hyperplasia (AAH). There were 39 cases of SBAC, 19 of NSBAC and 20 of AAH. To detect the loss of heterozygosity (LOH) we used the microsatellite markers D3S1234 and D3S1300 on chromosome 3p, IFNA and D9S144 on 9p, and TP53 on 17p. We also used polymerase chain reaction-SSCP analysis and direct sequencing to examine a point mutation of the p53 gene at exons 5-8. At the TP53 locus, the frequencies of LOH showed a statistical rank-difference correlation among AAH, NSBAC and SBAC. On chromosomes 3p and 9p there were no statistical differences of LOH among AAH, NSBAC and SBAC. We detected a significant statistical rank-difference correlation in the p53 mutation among AAH, NSBAC and SBAC. These findings suggest that a process of multistep carcinogenesis from AAH through NSBAC to SBAC might occur in some cases of adenocarcinoma, and LOH of 3p and 9p might be an early event of carcinogenesis, while the p53 mutation might be a later event.     

Comparative Ultrastructural Study of Atypical Adenomatous Hyperplasia and Adenocarcinoma of the Human Lung

Atypical adenomatous hyperplasia (AAH) of the human lung is considered a possible precursor of pulmonary adenocarcinoma. However, its true biological significance remains to be clarified. The authors studied the ultrastructure of AAH in surgically resected lungs and compared it with that of coexisting adenocarcinoma in an effort to define the characteristic features of AAH. Ultrastructurally, AAH possessed oval to irregular nuclei with high nucleo-cytoplasmic ratio and large nucleoli. Development of cytoplasmic organelles was generally poorer in AAH than in adenocarcinoma. However, these differences became less apparent as the degree of atypia of AAH advanced. Both lamellar bodies and electron-dense granules were found in AAH as well as in adenocarcinoma. These results suggest a close relation of AAH with adenocarcinoma of type 2 pneumocyte or Clara cell type. Further, the results of immunohistochemical studies for surfactant apoprotein A, urine protein 1, cytochrome P-450s, CEA, p53, c-erbB-2, Ki67, and bcl-2 well reflected the ultrastructural findings. These results suggest, in accordance with previous studies, that AAH is a lesion closely related to adenocarcinoma. Further, AAH shares some characteristics of type 2 pneumocytes and Clara cells, implying that it might be derived from their common precursor.     

Disproportionate representation of KRAS gene mutation in atypical adenomatous hyperplasia, but even distribution of EGFR gene mutation from preinvasive to invasive adenocarcinomas

In the resected lung, additional small lesions are occasionally found incidentally, and include the full spectrum of preinvasive to invasive lesions under the current putative schema of the sequential development of lung cancer. In this study, we examined EGFR and KRAS gene mutations in 119 synchronous pulmonary lesions, including 40 precursor lesions (atypical adenomatous hyperplasia, AAH), 26 carcinomas in situ (non-mucinous bronchioloalveolar carcinoma, BAC), 14 minimally invasive adenocarcinomas, 34 overt invasive adenocarcinomas, and five of other subtypes of cancer. Although the mutually exclusive nature of KRAS and EGFR gene mutations was maintained even in preinvasive lesions, the incidences of the lesions along the putative progression schema were quite different. The KRAS gene was mutated in 33% of AAH, 12% of carcinomas in situ, 8% of minimally invasive adenocarcinomas and 0% of well-differentiated adenocarcinomas, whereas the frequencies of EGFR mutation did not fluctuate greatly, at 25%, 51%, 36%, 86% and 67%, respectively. These results are consistent with the findings of a published gene-targeted mouse model; the mice expressing oncogenic KRAS developed AAH but not invasive adenocarcinoma, whereas a spectrum of preinvasive to invasive adenocarcinomas was observed in the mice expressing mutant EGFR. Taking these factors together, it is suggested that AAH could develop by either KRAS or EGFR gene mutation, but AAH harbouring a KRAS gene mutation might not progress further to an invasive cancer.     

Lung adenocarcinoma associated with atypical adenomatous hyperplasia. A clinicopathological study with special reference to smoking and cancer multiplicity

Atypical adenomatous hyperplasia (AAH) of the lung has been proposed as a possible precursor lesion of adenocarcinoma of the lung. In the present study, we sought to clarify the clinicopathological characteristics of lung adenocarcinoma cases associated with AAH, with special reference to tobacco smoking and the presence of multiple primary carcinomas of pulmonary and extrapulmonary organs. We examined 123 surgically resected lung adenocarcinomas and conducted histopathological diagnoses for AAH and multiple primary pulmonary carcinomas. Clinicopathological characteristics such as age, sex, smoking index, survival, and the presence of extrapulmonary primary carcinomas were obtained from clinical records, and the associations among these factors were examined statistically. Sixteen lung adenocarcinoma patients had accompanying AAH (the AAH group) and 107 cases did not (the NAAH group). The incidence of primary carcinomas in extrapulmonary organs was higher in the AAH group (37.5%; 6/16) than in the NAAH group (12.5%; 13/107) (P = 0.01). Multiple primary lung cancers tended to be more frequent in the AAH group, but the difference was not statistically significant (P = 0.07). Although there was no difference in tobacco smoking between the two groups, all eight cases with multiple primary lung carcinomas were smokers. Furthermore, multiple primary lung carcinomas were found more frequently in smokers of the AAH group (37.5%; 3/8) than in the smokers of the NAAH group (7.2%; 5/69) (P = 0.04). The results suggested that constitutional or genetic factors might predispose patients to the development of AAH together with extrapulmonary primary carcinomas, and that smoking might contribute to the development of multiple primary lung adenocarcinomas, especially in patients with pre-existing AAH.     

A case of double primary adenocarcinoma of the lung with multiple atypical adenomatous hyperplasia

A case of double primary adenocarclnoma of the lung with multiple atypical adenomatous hyperplasla (AAH) In a 77-year-old woman Is reported. Hlstopathologlcally, in the resected left upper lobe of the lung, both cancers were diagnosed as well-differentlated papillary adenocarcinoma, and 161 lesions of AAH were also found. Both the cancer lesions and six AAH (greater than 3 mm In diameter) were examined wlth regard to immunoreactivity of carcinoem-bryonlc antigen (CEA) and p53 gene product, microsatellite lnstabllity (MI) and loss of heterozygosity (LOH) on chromosome 9q and 17q by polymerase chain reaction (PCR). Although both cancers expressed CEA, they did not show clonal lmmunoreactivity for the p53 gene product. Atypical adenomatous hyperplasia expressed CEA weakly and showed no immunoreactlvity for p53 gene protein. Both carcinomas showed LOH on chromosome 17q, and one of them showed LOH on chromosome 9q. In six AAH, LOH on chromosome 17q was detected In two tumors, and one of them also showed LOH on chromosome 9q. One AAH, which was negative for LOH on chromosome 17q and 9q, showed Mi at D17S791. These results indicated that AAH is a clonal neoplastic lesion with genetic abnormalities and should be called intraepithelial pneumocyte neoplasia, and that each of the numerous papillary lesions in this case was considered to be an Independent lesion.     

Histological progression of small intrapulmonary metastatic tumor from primary lung adenocarcinoma

The histopathology of small metastases is thought to reflect the early metastatic process. To clarify the morphological features of early metastatic tumor progression, we analyzed the histological heterogeneity of many small intrapulmonary metastases. Histological typing based on the World Health Organization classification (bronchioloalveolar carcinoma, acinar, papillary, and solid subtype) was used to evaluate 234 metastases from the primary lung adenocarcinomas of 139 patients. The predominant subtype of metastasis 3 mm or less in diameter was bronchioloalveolar carcinoma when the primary lesion was diagnosed as predominant bronchioloalveolar carcinoma, acinar, and papillary subtype. When the histology of the primary tumor was predominantly a solid subtype, the predominant subtype of metastatic tumor was also a solid subtype. However, analysis of metastases that were more than 3 mm showed that the predominant subtype of the metastasis reflected the predominant subtype of the primary tumor. Furthermore, we evaluated the number of subtypes in primary and metastatic tumors. As the metastasis grew larger, the number of subtypes in the metastatic lesion increased and came close to the number composed in the primary lesion. These findings suggest that implanted cancer cells display lepidic growth in the early metastatic phase and recapitulate the morphological heterogeneity of the original tumor as the metastasis enlarges.     

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.     

Immunohistochemical study of Skp2 and Jab1, two key molecules in the degradation of P27, in lung adenocarcinoma

To clarify the association of the P27 degradation pathway proteins, Skp2 and Jab1, with the development and progression of lung adenocarcinoma (AD), we immunohistochemically investigated Skp2 and Jab1 expression together with P27- and Ki-67-labeling in 110 lung AD and 11 atypical adenomatous hyperplasia (AAH) and analyzed the relationship between the expression of these proteins and the clinicopathological factors. High Skp2 or Jab1 expression was frequent in lung AD (52/110, 47%, and 59/110, 54%, respectively), and high expression of Jab1 was also frequent in AAH (4/11, 36%), while it was not observed in normal bronchiolar epithelium. The P27 labeling index (LI) was reciprocally correlated with high Skp2 and Jab1 expression, and a higher Ki-67 LI was significantly correlated with high Skp2 and Jab1 expression. However, low P27 expression did not correlate with a higher Ki-67 LI. High Skp2 lung AD showed significant correlation with blood and lymphatic vessel invasion, which low P27 expression did not correlate with. Furthermore, high Skp2 expression in lung AD was significantly correlated with a poor outcome for patients. Thus, Skp2 and Jab1 regulate P27 degradation, and might contribute to the development and progression of lung AD through P27-mediated and -unmediated mechanisms.     

Loss of expression of E-cadherin and beta-catenin is associated with progression of pulmonary adenocarcinoma

The aim of the present study was to determine the association of loss of membranous expression of epithelial (E)-cadherin and beta-catenin with the progression of pulmonary adenocarcinoma. The expression of E-cadherin and beta-catenin was examined in 154 cases of pulmonary adenocarcinoma, including 49 cases of atypical adenomatous hyperplasia (AAH), 40 cases of bronchioloalveolar carcinoma (BAC), 42 cases of BAC-dominant type of adenocarcinoma with mixed subtypes (early MX) and 23 cases of BAC-recessive type of adenocarcinoma with mixed subtypes (overt MX), by immunohistochemistry. E-cadherin expression was positive in all cases of AAH, in 37 cases (92.5%) of BAC and in 34 cases (81.0%) of early MX, while it was positive in three cases (13.0%) of overt MX. beta-Catenin expression was positive in 47 cases (95.9%) of AAH, in 28 cases (70%) of BAC, in 32 cases (76.2%) of early MX and in 11 cases (47.8%) of overt MX. The rates of expression of E-cadherin and beta-catenin among cases of AAH, BAC, early MX and overt MX were significantly reduced. Loss of expression of E-cadherin and beta-catenin may play an important role in the progression of pulmonary adenocarcinoma, and these events occur before structural destruction of the alveolar wall by invasion of carcinoma cell.     

免疫组织化学和荧光原位杂交检测肺腺癌ROS1表达的对比

目的比较免疫组织化学(immunohistochemistry,IHC)和荧光原位杂交(fluorescent in situhybridization,FISH)技术在检测肺腺癌中ROS1蛋白表达和融合基因的差异性和一致性,评估IHC在临床上的应用价值。方法应用IHC和FISH技术对332例肺腺癌中ROS1蛋白表达和融合基因进行检测,比较两者检测的结果和相关性,并探讨ROS1融合基因与临床病理特征之间的关系。结果 332例肺腺癌中FISH阳性者13例,阴性者319例,ROS1融合基因阳性率为3.9%;ROS1蛋白表达结果:0者312例,1+者2例,2+者5例,3+者13例,ROS1蛋白过表达率为6.0%;经FISH检测312例ROS1蛋白表达为0的标本无ROS1融合基因,2例1+的标本中也无ROS1融合基因;5例2+的标本中有2例ROS1融合基因,13例3+的标本中11例有ROS1融合基因;IHC检测0和1+、2+、3+标本FISH检测阳性符合率分别为0(0/314)、40%(2/5)和84.6%(11/13);IHC检测ROS1蛋白为2+~3+的标本中72.2%(13/18)显示ROS1融合基因,0~1+患者中无1例有ROS1融合基因(Kappa系数=0.831,P0.01);IHC检测ROS1蛋白表达的敏感性和特异性分别100%和98.4%。结论 IHC检测ROS1蛋白表达和FISH检测ROS1融合基因,两种方法有较高的符合率,一致性较好,IHC有较高的敏感性和特异性,可作为临床检测肺腺癌ROS1融合基因的简单而有效的方法。     

Expression of Lewis X-related antigens in adenocarcinomas of lung

Fifty primary lung adenocarcinomas were examined immunohistochemically using monoclonal antibodies to determine changes in the expression of N-acetyl-lactosamine (blood group type-2 chain), Le^x, Le^Y and sialyl Le^x-i. These antigens were expressed in 60%, 70%, 90% and 94% of carcinomas, respectively; in 8%, 12%, 56% and 86% of normal broncho-bronchiolar epithelium; and in 32%, 0%, 100% and 0% of normal alveolar epithelium. The greater the complexity of the antigenic structure, the greater the incidence of positive staining in the adenocarcinomas. Although the more complex antigens such as sialyl Le^x-i and Le^Y have also been demonstrated in the sera of lung cancer patients, they were not always cancer-selective in our immunohistochemical study. In contrast, the less complex antigens such as N-acetyl-lactosamine (type-2 chain) and Le^x seem to be cancer-selective, as they showed low positivity in normal lung tissue.     

Chromosomal imbalances of primary and metastatic lung adenocarcinomas.

Comparative genomic hybridization (CGH) was used to screen 83 lung adenocarcinomas of 60 patients for chromosomal imbalances. The most common alteration was DNA overrepresentation on chromosome 1q, with a peak incidence at 1q22-q23 in 73% of the primary tumours, followed by DNA overrepresentation on chromosomes 8q and 20q, and deletions on chromosomes 3p, 4q, 6q, 9p, 9q, and 13q, in at least 60%. The generation of a difference histogram of metastasizing versus non-metastasizing tumours and a case-by-case histogram for the comparison of 23 paired samples of primary tumours and corresponding metastases suggested that deletions on chromosomes 3p12-p14, 3p22-p24, 4p13-15.1, 4q21-qter, 6q21-qter, 8p, 10q, 14q21, 17p12-p13, 20p12, and 21q, and overrepresentations on chromosomes 1q21-q25, 7q11.2, 9q34, 11q12-q13, 14q11-q13, and 17q25 are associated with the metastatic phenotype. In contrast, losses on chromosome 19 and gains on 3p, 4q, 5p, and 6q were preferentially found in non-metastasizing tumours. The analysis of the paired samples revealed considerable chromosomal instability, but indicated a clonal relationship in each case. The primary tumours often showed additional deletions, suggesting that loss of function mutations are critical in the initial phase of tumour dissemination, whereas the metastases preferentially acquired DNA gains, probably modulating the metastatic phenotype. The primary data from this study (ratio profiles, clinicopathological parameters, histograms) are also available at http://amba.charite.de/cgh.     

Ultrastructural heterogeneity of lung carcinomas: Representativity of samples for electron microscopy in tumor classification

Histologic heterogeneity of tumors is a well-known phenomenon, which has been repeated studied at the light microscopic level. Electron microscopy has been advocated as an adjunct in classification of tumors which pose difficulties on light microscopic classification. However, in view of tumor heterogeneity, it might be anticipated that the problem of sample error could detract from the usefulness of electron microscopy in tumor typing. Ultrastructural heterogeneity of tumors has thus far not been systematically investigated. We performed an ultrastructural randomized and blinded study of superficial and deep samples of 44 resected lung carcinomas, tumors which are notorious for their histologic heterogeneity. Neuroendocrine and squamous differentiation, as well as adenodifferentiation, were assessed separately and semiquantitatively in each sample. Twenty-six tumors showed more than one type of differentiation in at least one sample. However, in only two cases did the main type of differentiation differ between the two samples. A further nine cases showed one predominant differentiation type in both samples, but a similarly pronounced second differentiation type in one of the samples. Thus, in terms of ultrastructural diagnosis, the two samples showed a major discrepancy in two of 44 cases, and a minor discrepancy in nine of 44 cases. We conclude that ultrastructural heterogeneity of lung tumors is a common occurrence, but that it only rarely leads to totally different ultrastructural diagnoses.     

抗体鸡尾酒AMACR/P63/34βE12在前列腺良恶性病变鉴别诊断中的应用价值

目的:探讨抗体鸡尾酒AMACR/P63/34βE12在前列腺良恶性病变鉴别诊断中的应用价值。方法:收集2001~2005年111例前列腺手术切除标本，其中前列腺腺癌39例，高级别前列腺上皮内瘤（high-grade prostatic intraepithelial neoplasias，HGPIN）29例，非典型性腺瘤样增生（atypical adenomatous hyperplasia, AAH）3例，前列腺结节性增生（benign prostatic hyperplasia, BPH）40例。作抗体鸡尾酒AMACR/P63/34βE12的免疫标记，观察3种抗体在各类病变中的表达情况。结果:39例前列腺腺癌AMACR全部呈阳性，癌巢周围无基底细胞残存（P63/34βE12阴性）。29例高级别前列腺上皮内瘤变，14例（48.3%）腺泡上皮AMACR呈阳性，29例腺泡上皮周围有连续或不连续的基底细胞（P63/34βE12阳性）。3例非典型性腺瘤样增生中2例腺泡上皮AMACR呈弱阳性；3例腺泡上皮周围有较连续的基底细胞（P63/34βE12中度阳性）。40例前列腺结节性增生，腺泡上皮AMACR染色均呈阴性，周围有连续的基底细胞（P63/34βE12强阳性）。结论:鸡尾酒抗体AMACR/P63/34βE12标记前列腺组织，能够同时高特异性和敏感性地检测出前列腺腺癌细胞（或非典型增生的腺泡上皮细胞）和基底细胞，为前列腺腺癌与高级别上皮內瘤变、非典型性腺瘤样增生、前列腺结节性增生的鉴别诊断提供有力的证据。     

A metastatic signature in entire lung adenocarcinomas irrespective of morphological heterogeneity

Recent microarray expression studies support the hypothesis that metastatic potential is acquired early in tumorigenesis and that most tumor cells have the potential to metastasize. To assess this possibility, we investigated invasive lung adenocarcinomas, which characteristically display morphological heterogeneity with a less malignant appearance at the periphery as a model. In lymph node-positive lesions, gene expression profiles were compared among moderately differentiated components with an aggressive appearance, peripheral well-differentiated components with a less malignant appearance, and patient-matched lymph node metastases. We also compared these with node-negative lung adenocarcinomas, which are morphologically indistinguishable from node-positive tumors. Striking similarities were observed between pairs of primary and metastatic tumors, even within primary well-differentiated components. We generated a 75-gene signature separating primary lung adenocarcinomas according to lymph node status. Hierarchical clustering using this gene set identified a distinct independent group composed of node-positive cases, clearly separate from node-negative tumors and normal lung tissue. The results suggest that the metastatic signature is maintained throughout progression, implying that the entirety of a primary tumor, including the morphologically less malignant components, might have metastatic potential. This finding has profound clinical implications. In the future, the metastatic potential of tumors may be predicted by biopsy, helping to avoid unnecessary lymph node dissection in low-risk patients.     

Chromosomal numerical abnormalities in early stage lung adenocarcinoma

Chromosomal numerical abnormalities (CNA) are ubiquitous in human cancers. However, the question of when a CNA occurs in the course of tumor generation and progression, is controversial. Recent radiological scrutiny has enabled the identification of small peripheral lesions in the lung. A chromosome-wide investigation encompassing almost all the chromosomal centromeres was performed using modified fluorescence in situ hybridization on the archived pathological samples of 16 atypical adenomatous hyperplasia (AAH) and 30 lung adenocarcioma (AdCa) specimens including those smaller than 1 cm in size. The prevalence of the gain was more extensive in male than in female patients, and in non-smokers than in smokers. It tended to be greater in poorly differentiated AdCa, in moderately differentiated AdCa, and in well-differentiated AdCa cases, in that order. Most AAH had non-specific gains affecting all the examined chromosomes. The prevalence of the gain differed significantly between AAH and bronchioloalveolar carcinoma (BAC) 1 cm. It is proposed that the CNA is a distinct phenomenon occurring in the early or premalignant stage of lung AdCa, and that the CNA itself may not be a sequel in the carcinogenetic process, but a driving factor in carcinogenesis.