Wolter J. Mooi

K-ras Oncogene Activation as a Prognostic Marker in Adenocarcinoma of the Lung

BACKGROUND The capability of activated oncogenes to induce malignant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocarcinomas. METHODS We studied the clinical importance of this oncogene-activation in 69 patients with lung adenocarcinoma in whom complete resection of the tumor was possible. The polymerase chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation-specific oligonucleotide probes. RESULTS Nineteen of the tumors harbored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon-12 point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-ras point-mutation-positive tumors died during the follow-up period, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P less than 0.001). CONCLUSIONS The presence of K-ras point mutations defines a subgroup of patients with lung adenocarcinoma in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.

Mutational activation of the K-ras oncogene. A possible pathogenetic factor in adenocarcinoma of the lung.

To define the role of cellular oncogenes in human cancers, we studied the prevalence of mutational activation of ras oncogenes in untreated non-small-cell lung cancer. Genomic DNA was extracted from 39 tumor specimens obtained by thoracotomy and was examined for activating point mutations in codons 12, 13, and 61 of the H-ras, K-ras, and N-ras genes. A novel, highly sensitive assay based on oligonucleotide hybridization following an in vitro amplification step was employed. The K-ras gene was found to be activated by point mutations in codon 12 in 5 of 10 adenocarcinomas. Two of these tumors were less than 2 cm in size and had not metastasized. No ras gene mutations were observed in 15 squamous-cell carcinomas, 10 large-cell carcinomas, 1 carcinoid, 2 metastatic adenocarcinomas from primary tumors outside the lung, and 1 small-cell carcinoma. An approximately 20-fold amplification of the unmutated K-ras gene was observed in a tumor that proved to be a solitary lung metastasis of a rectal carcinoma. We conclude that mutational K-ras activation may be an important early event in the pathogenesis of adenocarcinoma of the lung but that amplification of ras genes or mutational activation of H-ras or N-ras does not play a major part in non-small-cell lung cancer.

Losses of Chromosomes 1p and 3q Are Early Genetic Events in the Development of Sporadic Pheochromocytomas

Despite several loss of heterozygosity studies, a comprehensive genomic survey of pheochromocytomas is still lacking. To identify DNA copy number changes which might be important in tumor development and progression and which may have diagnostic utility, we evaluated genetic aberrations in 29 sporadic adrenal and extra-adrenal pheochromocytomas (19 clinically benign tumors and 10 malignant lesions). Comparative genomic hybridization was performed using directly fluorochrome-conjugated DNA extracted from frozen (16) and paraffin-embedded (13) tumor tissues. The most frequently observed changes were losses of chromosomes 1p11-p32 (86%), 3q (52%), 6q (34%), 3p, 17p (31% each), 11q (28%), and gains of chromosomes 9q (38%) and 17q (31%). No amplification was identified and no difference between adrenal and extra-adrenal tumors was detected. Progression to malignant tumors was strongly associated with deletions of chromosome 6q (60% versus 21% in clinically benign lesions, P = 0.0368) and 17p (50% versus 21%). Fluorescence in situ hybridization confirmed the comparative genomic hybridization data of chromosomes 1p, 3q, and 6q, and revealed aneuploidy in some tumors. Our results suggest that the development of pheochromocytomas is associated with specific genomic aberrations, such as losses of 1p, 3q, and 6q and gains of 9q and 17q. In particular, tumor suppressor genes on chromosomes 1p and 3q may be involved in early tumorigenesis, and deletions of chromosomes 6q and 17p in progression to malignancy.

Enhanced expression of vascular endothelial growth factor in lungs of newborn infants with congenital diaphragmatic hernia and pulmonary hypertension

BACKGROUND Pulmonary hypoplasia accompanied by pulmonary hypertension resistant to treatment is an important feature of congenital diaphragmatic hernia (CDH). The pathogenesis of the pulmonary vascular abnormalities in CDH remains to be elucidated at the molecular level. Vascular endothelial growth factor (VEGF), an endothelial cell specific mitogen, is known to play a role in pulmonary angiogenesis and vascular remodelling but there are no data on VEGF expression in patients with CDH. METHODS Necroscopic lung specimens from 21 patients with CDH with lung hypoplasia and from seven age matched control newborn infants without lung hypoplasia were processed for immunohistochemical analysis using affinity purified anti-human VEGF antibodies. All the cases of CDH had pulmonary hypoplasia, indicated by a lung/body weight index of ⩽0.012, and pulmonary hypertension indicated by repeated cardiac ultrasonography. Cellular localisation of VEGF was semiquantitatively analysed using a staining score ranging from 0 (no staining) to 4 (very strong staining). RESULTS Significantly raised levels of VEGF immunoreactivity were observed in lung specimens from cases of CDH compared with controls. VEGF was detected mainly in the bronchial epithelium and the medial smooth muscle cells of large (>200 μm) and small (<200 μm) pulmonary arteries, the most intense staining being in the medial smooth muscle cells of the small pulmonary arteries. Endothelial cells were positive for VEGF staining in patients with CDH but not in controls. CONCLUSIONS This is the first study of VEGF expression in newborn infants with CDH. Increased levels of VEGF, especially in the small, pressure regulating pulmonary arteries, point to a potential role in vascular remodelling. This may reflect an unsuccessful attempt by the developing fetus to increase the pulmonary vascular bed in the hypoplastic lungs to alleviate the associated pulmonary hypertension.

Enhanced expression of vascular endothelial growth factor in pulmonary plexogenic arteriopathy due to congenital heart disease

Congenital heart disease (CHD) leading to increased pulmonary blood pressure and flow is an important cause of pulmonary plexogenic arteriopathy (PPA). This type of arteriopathy tends to progress to an irreversible stage, hallmarked histologically by the emergence of a number of characteristic lesions, which include concentric laminar intimal proliferation and fibrosis, and plexiform lesions. The pathogenesis of these lesions, which connote a very poor prognosis, is not well understood. Since endothelial cell proliferation has been demonstrated in these lesions, it was hypothesized that vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, might play a role in their pathogenesis. Thirty‐nine patients with various types of CHD, who underwent cardiac catheterization and subsequent cardiac surgery, were studied prospectively. On the basis of a detailed assessment of the type of cardiac defect, the haemodynamic abnormalities, and the histopathological features evident from open lung biopsies, taken in all instances, patients were histologically grouped into cases with moderate PPA (n=18), advanced PPA (n=7), pulmonary congestive vasculopathy (PCV, n=5), and controls lacking pulmonary hypertension or increased pulmonary blood flow (n=4). Five patients were excluded from analysis because of inadequate sample size or quality. The presence of VEGF was assessed immunohistochemically using standard procedures and was correlated with haemodynamic and histological data. Immunoreactive VEGF was detected in pulmonary arterial smooth muscle cells and endothelial cells in 13 out of 34 cases and was more frequent and more pronounced in patients with the histological lesions of advanced PPA than in those with moderate PPA (p<0.01). VEGF positivity was particularly prominent in the lesions characteristic of advanced PPA. No difference in VEGF expression was observed between controls, PVC, and moderate PPA cases. Measured haemodynamic parameters did not differ significantly between VEGF‐positive and VEGF‐negative cases. We conclude that VEGF may play a role in the angioproliferative changes of advanced PPA. Copyright

Impaired structural remodelling of pulmonary arteries in newborns with congenital diaphragmatic hernia: a histological study of 29 cases

Congenital diaphragmatic hernia (CDH) is associated in many cases with lung hypoplasia and pulmonary hypertension (PH). The pathogenetic mechanisms underlying the pulmonary hypertension in CDH are not completely understood. In order to alleviate the pulmonary hypertension, new therapeutic modalities have been introduced including extracorporeal membrane oxygenation (ECMO). This paper reports a study of the histology of the lungs of 29 CDH autopsy cases, with special attention to the pulmonary arteries, and relating the findings to gestational age and ECMO treatment. Formalin‐fixed and paraffin‐embedded specimens were stained with haematoxylin and eosin (H&E) and elastic van Gieson (EvG) stains, followed by morphometric measurements of the arterial media and adventitia. As expected, there was a significant decrease in adventitial percentage and total wall thicknesses of small pulmonary arteries with an external diameter less than or equal to 150 µm in term control newborns compared with pre‐term controls ( p=0·0004 and 0·05). In CDH newborns, all the measured values of the arterial wall remained significantly higher. The increase of adventitial thickness also affected the supernumerary arteries in CDH neonates. CDH newborns subjected to ECMO treatment showed a significantly thinner arterial adventitia than CDH cases who did not receive ECMO ( p=0·0001), the former approaching normal values. These results indicate that in CDH, there is failure of the normal arterial remodelling processes occurring in the perinatal period. The adventitial thickening, which has been reported previously in term CDH patients only, was related in the present study to differences in gestational ages. This appears to be partially reversed by ECMO treatment, thus constituting one of the mechanisms by which ECMO treatment aids in alleviating the associated PH in CDH newborns. Copyright

Clear cell sarcoma of tendons and aponeuroses (malignant melanoma of soft parts) and cutaneous melanoma: exploring the histogenetic relationship between these two clinicopathological entities

Clear cell sarcoma of tendons and aponeuroses (CCS) has come to be recognized as a distinct histopathological entity in the last three to four decades. It shares a number of histological and ultrastructural features with cutaneous melanoma (MM), occasionally creating diagnostic difficulties with metastatic melanoma in the absence of a known primary cutaneous tumour. At a genetic level, a t(12;22) has been identified in 60–75 per cent of cases of CCS using karyotype analysis, while MM demonstrates a broad range of genetic alterations, most commonly appearing to involve chromosomes 1, 5, and 6. Although these two tumour types share many common microscopic, and thus histogenetic, similarities, the genotypic evidence supports two distinct histopathological entities.

A Rapid and Simple Procedure for the Routine Detection of ras Point Mutations in Formalin-Fixed, Paraffin-Embedded Tissues

The use of the polymerase chain reaction (PCR) to detect specific DNA sequences in small amounts of tissues or cells has become a widespread tool in the field of molecular biology. With the better understanding of the clinical significance of oncogene activations in human tumors, the application of PCR in a routine setting is rapidly gaining importance. We have developed a rapid and simple procedure for the detection of mutated ras oncogenes in routinely fixed, paraffin-embedded tissue samples. DNA is isolated from three 10 μm tissue sections by incubation with a nonionic detergent and proteinase K, and can be directly used for amplification by PCR. The amplified DNA fragments are then dot-blotted onto nylon membranes and are hybridized to radioactively labeled oligodeoxynucleotides, specific for each of the mutated ras sequences. After a selective washing procedure, only fully matched oligodeoxynucleotides remain bound to the membrane, thus revealing the nature of the sequences that were present in the starting material. With this method, the detection of point mutations in ras genes can be performed in a routine setting, and the results of the analyses can be available in as few as 3–4 days.

Pathological features of glycogen storage disease type II highlighted in the knockout mouse model

Glycogen storage disease type II (GSDII; Pompes disease) is an autosomal recessive disease caused by lysosomal α‐glucosidase deficiency. Skeletal muscle weakness is the most conspicuous clinical symptom of patients suffering from GSDII and skeletal muscle also is prominently involved in the knockout mouse model of this disease. Thus far, however, little detailed information has been published on the pathological changes in other mouse tissues. This paper aims to provide these data and gives a record of the clinical course of the mouse model over a 2‐year period. Four‐month‐old affected mice perform worse in a running wheel than their unaffected littermates, but do not yet display other clear signs of disease. The lysosomal glycogen storage, already evident at birth, becomes more severe in time, leading to muscle wasting by 9–10 months of age and then limb girdle weakness and kyphosis. The disease does not markedly shorten the animals life span despite the serious tissue pathology, which is not limited to heart and skeletal muscle, but is also seen in the smooth muscle of blood vessels and of the respiratory, digestive, and urogenital tracts. In addition, the mice have lysosomal glycogen storage in the liver, kidney, spleen, and salivary gland; in Schwann cells of the peripheral nerves, and in a subset of neurons in the central nervous system. By pathological criteria, the knockout mouse model parallels the human infantile form of GSDII and is attractive for studying the possible reversal of tissue pathology and symptomatology under different therapeutic regimes. Copyright

Pulmonary hypertension in human newborns with congenital diaphragmatic hernia is associated with decreased vascular expression of nitric-oxide synthase

The molecular basis of the pathogenesis of pulmonary hypertension (PH) associated with congenital diaphragmatic hernia (CDH) is poorly understood. Variation in responses to therapeutic strategies such as nitric oxide (NO) inhalation and extracorporeal membrane oxygenation (ECMO) in patients with CDH remains a major problem in pediatric critical care. We investigated the expression pattern of NO-generating enzyme nitricoxide synthase (NOS) (both endothelial [eNOS] and inducible [iNOS] isoforms) in the lungs of CDH patients with PH and evaluated the influence of ECMO on the expression levels of these genes in an attempt to understand the underlying molecular mechanisms. Lung autopsy specimens from 23 cases of CDH not treated by ECMO and 10 ECMO-treated CDH cases were studied and compared with 11 age-matched controls. Expression of iNOS and eNOS was assessed by immunohistochemistry and video-image analysis. Expression of iNOS in the endothelium of small pulmonary arteries (external diameter≤200 μm) was significantly lower in CDH cases that had not received ECMO treatment (p=0.04). ECMO-treated CDH cases did not differ from controls in iNOS expression. Alveclar macrophages (CD68+ cells), of which the number also was increased, showed significantly enhanced staining for iNOS in CDH cases (p=0.03) compared with controls. The observed decrease in pulmonary expression of iNOS in patients with CDH suggests a potential role in the pathogenesis of pulmonary hypertension in newborns with CDH. ECMO treatment was correlated with induction of this enzyme, which may result in NO-mediated vasodilatation and thereby transiently reduce the pulmonary hypertension in CDH.