Radiol Oncol 2006; 40(Suppl 1): S67-S76. Detection of early lung cancer lesions in surgical resections and in bronchial and transbronchial biopsies Odkrivanje zgodnjih oblik pljučnega raka v kirurških resektatih in bronhialnih in transbronhialnih biopsijah Tomaž Rott1, Maja Jerše1, Marjeta Terčelj2, Janez Eržen3 1Institute of Pathology, Medical Faculty, University of Ljubljana, 2Centre of Pulmonary Diseases and Allergy, and 3Clinical Department of Thoracic Surgery, Clinical Centre Ljubljana, Slovenia Background. Overall bad prognosis of lung cancer is mostly due to too late detection of early lung cancer, which may be treated with good success. Therefore, different diagnostic methods are developing for more efficient detection of early lung cancer: besides modern radiological, bronchoscopic methods with additional fluorescence techniques, quantitative cytological investigations, also histological and molecular investi-gations are included. Histology may reveal early preinvasive lung cancer lesions, associated early during multistep lung carcinogenesis with molecular genetic changes. Patients and methods. Preinvasive epithelial lung cancer lesions we searched in two groups of patients. In the first group of 316 patients from the period March 2003 – August 2006, 498 bronchial and transbronchial biopsies were examined for squamous metaplasia and dysplasia, carcinoma in situ, and invasive tumours. In the second group of 238 patients from the period January 2004 - August 2006, resected primary lung tumours were analysed for preinvasive and invasive neuroendocrine tumours and atypical adenomatous hyperplasia. Results. The most frequent changes in bronchial and transbronchial biopsies were squamous metaplasia (46.5%), simple or goblet cell hyperplasia of the bronchial epithelium (44.3%), malignant tumours (20.66%) and squamous dysplasia (16.1%), but rare carcinoma in situ (0.63%). Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia was found in 15 (6.3%) cases in the vicinity of 238 resected lung cancer specimens, carcinoid in 12 patients (5%), and mostly combined large cell neuro-endocrine cancer in 21 patients (8.8%). Atypical adenomatous hyperplasia was found in 2 patients. Conclusions. Classical histological analysis should be focused on detection of early preinvasive epithelial lung cancer lesions. Additional available molecular investigations may reveal gradual genetic changes cha-racteristic for a series of the preinvasive epithelial histological changes. More systematically oriented sam-pling of lung resections could increase the incidence of the lesions, arising from pulmonary neuroendocrine cells, and especially the incidence of atypical adenomatous hyperplasia. Key words: lung neoplasms – pathology; metaplasia; hyperplasia; carcinoma in situ; biopsy; preinvasive epithelial lesions; light-microscopy S68 Rott T et al / Pathohistology and detection of early lung cancer Izhodišča. V celoti slaba napoved preživetja pljučnega raka je v večji meri posledica poznega odkrivanja te bolezni, saj le zgodnje oblike lahko uspešno zdravimo. Zato danes razvijamo različne diagnostične metode, s katerimi bi lahko uspešneje odkrivali zgodnje oblike pljučnega rak: Poleg modernih radioloških, novejših bronhoskopskih z dodatnimi fluorescenčnimi metodami, kvantitativnih citoloških preiskav tudi histološke in molekularne. Histologija lahko odkrije predinvazivne oblike pljučnega raka, ki jih že zgodaj na določenih stopnjah kancerogeneze spremljajo določene molekularne oziroma genetske spremembe. Bolniki in metode. Predinvazivne epitelne oblike pljučnega raka so iskali pri dveh skupinah bolnikov. V prvi skupini 316 bolnikov iz obdobja od marca 2003 do avgusta 2006 so v 498 bronhialnih in transbronhialnih biopsijah iskali predvsem ploščatocelično metaplazijo in displazijo, carcinoma in situ in invazivne tumorje. V drugi skupini 238 bolnikov iz obdobja od januarja 2004 do avgusta 2006, ki so jim kirurško odstranili primarne pljučne tumorje, pa so iskali predvsem predinvazivne in invazivne nevroendokrine tumorje in atipično adenomatozno hiperplazijo. Rezultati. Najpogostejše spremembe v bronhialnih in transbronhialnih biopsijah so bile ploščatocelična me-taplazija (46,5%), enostavna ali mukocelularna hiperplazija bronhialnega epitelija (44,3%), maligni tumorji (20,66%) in displazija (16,1%), redko je bil odkrit carcinoma in situ (0,63%). V pljučnih resektatih je bila difuzna idiopatska hiperplazija pljučnih nevroendokrinih celic odkrita pri 15 (6,3%), karcinoid pri 12 (5%) in pretežno kombinirani velikocelični nevroendokrini karcinom pri 21 bolnikih (8,8%). Atipična adenomatozna hiperplazija je bila prisotna pri 2 bolnikih. Zaključki. Klasični histološki pregled mora biti osredotočen na odkrivanje zgodnjih predinvazivnih epitelnih oblik pljučnega raka. Dodatne razpoložljive molekularne preiskave pa lahko odkrivajo postopne genetske spremembe, ki so značilne za postopne predinvazivne histološke spremembe v epiteliju. Bolj usmerjeno vzorčenje resektatov bi verjetno povečalo incidenco patoloških sprememb, ki izvirajo iz nevroendokrinih celic, predvsem pa incidenco atipične adenomatozne hiperplazije. Ključne besede: pljuča novotvorbe – patologija; metaplazija; hiperplazija; karcinom in situ; biopsija; pre-dinvazivne epitelne spremembe; svetlobna mikroskopija Introduction Lung cancer is the most important cancer in men and one of the most frequent also in women. Among all the patients with cancer, it is the leading cause of death, and it exceeds the sum of deaths due to cancer of breast, colon, and prostate.1 The survival rate is still very poor, mostly because of too late detection of the cancer in advanced stages, in more than two thirds of cases. But on the other hand, Stage I patients may have a 5-year survival approaching 70%.1,2 Correspondence to: Prof. Tomaž Rott, MD, PhD, Institute of pathology, Faculty of Medicine, Korytkova 2, 1000 Ljubljana, Phone: + 386 1 543 7100; Fax: + 386 1 543 7101; E-mail: tomaz1945@yahoo.com Therefore, the extreme effort is dedicated to its early detection by more complicated radiological, bronchoscopical, cytological, and molecular investigations. And finally, the classical histological analysis of dia-gnostic specimens and surgical resections should concentrate on the detection of precancerous preinvasive epithelial lesions, defined also by the last WHO classification of the lung tumours: squamous dysplasia, carcinoma in situ, diffuse idiopathic pul-monary neuroendocrine cell hyperplasia, and atypical adenomatous hyperplasia.3 Therefore, the aim of the study was to find out the incidence of preinvasive lesions in our routine diagnostic bronchial and transbronchial biopsies and in surgical spe-cimens. Radiol Oncol 2006; 40(Suppl 1): S67-S76. Rott T et al / Pathohistology and detection of early lung cancer S69 Table 1. Histological findings in 316 patients with 498 bronchial and transbronchial specimens Histological finding Number of patients Number of specimens EPITHELIAL HYPERPLASIA SIMPLE HYPERPLASIA GOBLET CELL HYPERPLASIA 140 (44.3%) 87 (27.5%) 73 (23.1%) 191 (38.3%) 101 (20.3%) 90 (18%) SQUAMOUS METAPLASIA 147 (46.5%) 188 (37.7%) ANGIOGENIC SQUAMOUS DYSPLASIA (MICROPAPILLOMATOSIS) 3 (0.95%) 3 (0.6%) BRONCHIAL EPITHELIAL DYSPLASIA 51 (16.1%) 59 (11.8%) CARCINOMA IN SITU 2 (0.63%) 3 (0.6%) BENIGN TUMOURS 4 (1.26%) 4 (0.8%) INVASIVE CARCINOMAS SQUAMOUS CARCINOMA ADENOCARCINOMA LARGE CELL CARCINOMA SMALL CELL CARCINOMA CARCINOID NOT SPECIFIED SUSPECTED MALIGNANCY 66 (20.88%) 16 16 15 12 1 1 5 78 (15.7%) NOT DIAGNOSTIC TISSUE 32 (10.12%) 38 (7.6%) Patients and methods There were 498 bronchial and transbronchial biopsies performed in 316 patients. The biopsies represent a part of random diagno-stic biopsies from the period March 2003 – August 2006. The patients demonstrated various mostly not specific symptoms and signs of lung involvement because of some not yet determined pathological process. Standard routine stains of the histological slices included haematoxylin and eosin, van Gieson-Weigert staining for elastin and collagen, alcian blue staining for eventual mucinogenesis and Perls’ staining to de-monstrate haemosiderin deposition. The specimens were thoroughly examined for changes especially in the bronchial epithe-lium and in associated lung tissue. Moreover, 238 patients with primary lung tumours, from the period january 2004-august 2006, are included in the stu-dy. They were treated surgically with lo-bectomy, bilobectomy or even pulmectomy together with the resection of regional lymph-nodes. Resected lobes or lungs with pulmonary tumour were sampled for the further histological examination. Besides routine hematoxylin and eosin stain, com-bined van Gieson-Weigert staining, alcian blue staining, some additional immune-histological stainings were employed. In all cases, marker for chromogranin A was used to demonstrate eventual neuroen-docrine component of the tumour, inclu-ding hyperplasia of neuroendocrine cells in the bronchial walls in the vicinity of the tumours. In majority of cases, thyroid transcription factor 1 (TTF-1) was used to confirm Radiol Oncol 2006; 40(Suppl 1): S67-S76. S70 Rott T et al / Pathohistology and detection of early lung cancer Figure 1. Simple epithelial hyperplasia with pseudos-tratification, haematoxylin and eosin. Figure 2. Combined superficial goblet cell hyperplasia and deep early squamous metaplasia, alcian blue. most probable pulmonary origin of adeno-carcinomas. In some cases, other additional immune-histological methods were used. Results Bronchial and transbronchial biopsies Bronchial and transbronchial biopsies di-splayed a wide spectrum of histological changes in bronchial epithelium presented in Table 1. Individual changes may occur simultaneously, especially if the tissue spe-cimen is abundant; in such cases, simple epithelial hyperplasia (with pseudostratifi-cation) is associated with goblet cell hyper- plasia, focal squamous metaplasia and even squamous dysplasia. Insufficient or inappropriate specimens pre-sented mostly bronchial fibrous tissue with inflammatory changes, without any epithe-lium. Dilated mucous glands and ducts are the changes suggesting chronic catarrhal bronchitis. In the lung, there is only focal fibrosis or not specific inflammatory chan-ges, without any bronchiolar or alveolar epithelium. Such unsatisfactory biopsies occurred in 32 patients, with mostly only one tissue specimen. Epithelial hyperplasia was presented by focal or diffuse simple hyperplasia (Figure 1), or by focal or diffuse goblet cell hyperplasia. Table 2. Interrelations between simple epithelial hyperplasia, squamous metaplasia and 59 cases of dysplasia. DIFFUSE SQUAMOUS METAPLASIA FOCAL SQUAMOUS METAPLASIA DIFFUSE SIMPLE HYPERPLASIA OTHER ASSOCIATED LESIONS WITH DYSPLASIA DYSPLASIA I 26 patients 11 patients 2 patients 2 (1 fibrosis and 1 adenocarcinoma) DYSPLASIA II 6 patients 8 patients 1 patient 1 bronchial fibrosis DYSPLASIA III 0 1 patient 0 1 carcinoma in situ Radiol Oncol 2006; 40(Suppl 1): S67-S76. Rott T et al / Pathohistology and detection of early lung cancer S71 Figure 3. Moderate squamous dysplasia with numer-ous mitoses in the lower part of the epithelium, hae-matoxylin and eosin. In many cases, the latter may cover under-lying early squamous hyperplasia represented by cells with obvious intercellular bridges (Figure 2). Developed squamous metaplasia affects the whole width of epithelium, and may be focal or diffuse, with hyperplastic or also with atrophic epithelium. Dysplastic changes are mostly associated with developed focal or diffuse squamous metaplasia, but only in rare cases with simple epithelial hyperplasia (Figures 3, 4). They may be found in the vicinity of an intraepi-thelial or invasive carcinoma (Table 2). A special form of relatively rare but un-favourable preinvasive dysplastic lesions is Figure 5. Angiogenic squamous dysplasia “micropapil-lomatosis” with vascular budding in the epithelium, haematoxylin and eosin. Figure 4. High-grade squamous metaplasia with mi-toses in the upper and lower thirds of epithelium, haematoxylin and eosin. angiogenic squamous dysplasia, with charac-teristic vascular budding into epithelium, previously reported as micropapillomatosis (Figure 5). Benign tumours included 2 bronchial squamous papillomas, endobronchial ha-martoma and submucous leiomyoma. The relative incidence of malignant tumo-urs is such as expected, with predominance of squamous and adenocarcinoma, follo-wed by small cell carcinoma. A relatively high incidence of large cell carcinoma is mostly due to insufficient and also dama-ged tumorous tissue. Suspected malignan-cies were characterized by keratin masses Figure 6. Discrete unrecognizable diffuse idiopathic pulmonary neuroendocrine cell hyperplasia in the epithelium and tumorlet, haematoxylin and eosin. Radiol Oncol 2006; 40(Suppl 1): S67-S76. S72 Rott T et al / Pathohistology and detection of early lung cancer Table 3. Neuroendocrine (NE) lesions among 238 surgically resected lung specimens Lesion Number of patients and % DIFFUSE IDIOPATHIC PULMONARY NE CELL HYPERPLASIA (DIPNEH) ASSOCIATED LESIONS DIPNEH + TUMORLET + CARCINOID DIPNEH + TUMORLET + CARCINOID + BRONCHIOLO-ALVEOLAR ADENOCARCINOMA DIPNEH + CARCINOID DIPNEH + LARGE CELL NE CARCINOMA + PLEOMORPHIC CARCINOMA DIPNEH + ADENOCARCINOMA DIPNEH + SQUAMOUS CELL CARCINOMA (1 BIFOCAL) DIPNEH + COMBINED ADENO-SQUAMOUS CARCINOMA 15 (6.3%) 1 1 2 1 6 3 1 CARCINOID 12 (5%) LARGE CELL NE CARCINOMA COMBINED LARGE CELL NE CARCINOMA 21 (8.8%) 14 (5.9%) without vital tissue, or by small nests of tumorous tissue, or by abundant necrotic masses with solitary cancer cells. Surgical resections Neuroendocrine lesions in surgical speci-mens are demonstrated in Table 3. Diffuse idiopathic atypical hyperplasia of the lung neuroendocrine cells was found in 15 (6.3%) cases of 238 resected lung specimens, from discrete to larger nodules of neuroendocrine cells. It was associated with various neuroendocrine tumours, with 2 tumorlets (Figures 6, 7) with 3 carcino-ids, and with 1 large cell neuroendocrine carcinoma, but also with not-neuroendocri-ne tumours, 6 adenocarcinomas (47%), 3 squamous carcinomas (one of them bi-focal), and 1 combined adenosquamous carcinoma (which demanded pulmonary resections). Radiol Oncol 2006; 40(Suppl 1): S67-S76. Carcinoid was found in 12 of 238 patients (5%). Carcinomas with neuroendocrine component (in 2-100% of all tumour cells) were found in 21 of 238 patients (8.8%), out of them, there were 14 cases of combined large cell neuroendocrine cancer (with elements of squamous cell carcinoma or adenocarci-noma). Only in 2 patients of 238 lung resecti-ons, atypical alveolar hyperplasia leading to peripheral adenocarcinoma was found at the periphery of papillary adenocarcino-mas (Figure 8). Reactive cuboid metaplasia (pneumocyte II hyperplasia) may be fo-und around almost all inflammatory and malignant neoplastic lesions, irrespective of carcinoma type. Cuboid metaplasia, to-gether with endogenic lipid pneumonia, suggest tissue destruction, usually found in malignant tumours or necrotic inflammato-ra granulomatous lesions. Rott T et al / Pathohistology and detection of early lung cancer S73 Figure 7. Small nests of hyperplastic epithelial neu-roendocrine cells, presenting early diffuse idiopathic pulmonary neuroendocrine cell hyperplasia, and nests of tumorlet in bronchial wall, chromogranin A. Discussion The extreme effort is dedicated to an early detection of preinvasive or microinvasive lung cancer lesions by more complica-ted radiological examinations (e.g. low-dose spiral computer tomography),4 auto-fluorescence bronchoscopy,5 fluorescence bronchoscopy with the additional spectral analysis,6,7 quantitative cytology8,9 and molecular investigations.10 ,11 And finally, also a classical histological analysis of dia-gnostic specimens and surgical resections should concentrate on the detection of precancerous preinvasive epithelial lesions, defined also by the last WHO classification of the lung tumours and mentioned in the introduction.3 Squamous metaplasia, dysplasia and in-traepithelial cancer, leading to invasive squamous carcinoma, to large cell carcino-ma, and to at least a great part of cases with pleomorphic cancer, are relatively frequent in patients with higher risk for the development of cancer (such as are also long-term smokers aged over 50 years). Less frequent is diffuse idiopathic pul-monary neuroendocrine cells hyperplasia, leading to neuroendocrine tumours, such as are tumorlet, typical and atypical car- Figure 8. Atypical adenomatous hyperplasia, haema-toxylin and eosin. cinoid, and probably to a part of large cell neuroendocrine carcinomas. There is not always a sharp demarcation between reactive cuboid metaplasia and atypical adenomatous hyperplasia, therefo-re, the incidence of the latter is in a relative wide range. Carcinogenesis of the lung cancer is a gradual multi-step process at both pheno-typic and genetic levels, similar to those found in other organic systems. Histologic (phenotypic) preinvasive bronchial lesions are usually multifocal, and are preceded or accompanied by multi-step accumulation of molecular and genetic abnormalities. Molecular changes are very rare in normal epithelium, few in hyperplasia, but numerous in dysplasia and in carcinoma in situ.12 Ten to twenty molecular changes are associated with progressive reversible (hyperplasia, metaplasia, mild dysplasia) or irreversibile (severe dysplasia, carcino-ma in situ, invasive carcinoma) histological changes (Table 4). Early histological changes in bronchial and alveolar epithelium leading to lung carcinoma may be observed and followed, altogether with associated gradual genetic changes. The analysis of molecular gene-Radiol Oncol 2006; 40(Suppl 1): S67-S76. S74 Rott T et al / Pathohistology and detection of early lung cancer Table 4. Chronology and frequency of genotypic and phenotypic-histologic changes during multi-step pathogenesis of squamous cell lung cancer (LOH- loss of heterozygosity, MSI-microsatellite instability, modified from Brambilla 2005) Norma hyperplasia metaplasia dysplasia carcinoma in situ (epithelium) Frequency -----------------------------------------------------------methylation-------------------------- 100% MSI----------------------------------------------------------------------------------------------- 50% 3p21.3 - LOH, small telomeric deletions----------------------------contiguous deletions 80% 9p21-CDKN2 - LOH------------------------------------------------------------------------- 70% 17p - LOH--------------------------------------------------------------------------------------- MYC overexpression--------------------------------------------------------- 60% ?-----------------telomerase dysregulation-------------telomerase upregulation 80% ?-----------------P53 + mutation---------------------------------------------- 70% ?---------------------------------------Aneuploidy-------------------------- 80% ?------------------FHIT ------------------------------------------------------------------------- 40% cyclins +-------------------------------------------------- bcl2>bax-------------------------------------------------- 8p21-23 - LOH------------------------------------------- 80% P16INK4----------------------------- K-RAS mutation-------------------- 20% Neoangiogenesis------------------- 40% 5q21-APC-MCC-LOH 30% RB----------------- tic changes requires greater technical and financial demands, although some investi-gations are relatively easily performed by immune-histochemical methods. Changes in the bronchial epithelium are very frequent, associated with various out-door irritants, including infections, smoking etc. They may be solitary but mostly multiple, synchronous or metachronous, in different parts of the lung. Therefore, we can expect synchronous and metachronous lung tumours, which may appear in more than 10%, also based on our data. Together with the tumours of the upper respiratory system, which is exposed to similar or same noxious agents, the incidence of their mul-tiplicity raises to 30%. Goblet cell hyperplasia should be menti-oned because of its role in etiopathogenesis of squamous metaplasia development. It is obvious that developed squamous meta-Radiol Oncol 2006; 40(Suppl 1): S67-S76. plasia may derive either through basal cell hyperplasia or more frequently through goblet cell hyperplasia. In many cases, we have seen the goblet cell hyperplasia »un-dermined« with early squamous metaplasi-as with obvious intercellular bridges. Dysplastic changes occur mostly in pati-ents with diffuse or focal squamous metapla-sia, and only in rare instances with diffuse epithelial hyperplasia. The follow-up of the patients with diffuse squamous metaplasia is therefore reasonable. Moreover, advanced dysplastic changes should be evaluated wi-th great concern and care, especially if they are found in different dislocated regions of the lung. The use of proliferation markers or genetic molecular analysis is advisable to find out the high risk patients. Very recently, there was a report about surviving, an inhibitor of apoptosis protein, which is absent in the normal epithelium, Rott T et al / Pathohistology and detection of early lung cancer S75 non-neoplastic lung parenchyma adjacent to tumour, and in the low-grade atypical adenomatous hyperplasia. But it is expres-sed in the areas of squamous metaplasia and dysplasia as well as in the high grade atypical adenomatous hyperplasia adjacent to the tumour.13 Very promising marker, if it will be proved true! Dysplastic changes in the vicinity of foreign bodies, or caused by long-lasting mycotic infections probably exhibit lower proliferative capacity in comparison wi-th dysplastic changes leading to invasive cancer or in the vicinity of developed lung cancer (Rott, personal unpublished obser-vations). Therefore, in dubious cases, the use of markers of proliferative activity is recommended. What is the reason for low incidence of intraepithelial carcinoma, carcinoma in situ? Does intraepithelial carcinoma rapidly change to invasive carcinoma? It is more likely that the undervaluation of dysplastic changes, which are at least in some cases de facto intraepithelial carcinoma, is the cause. Diffuse idiopathic pulmonary neuroen-docrine cells hyperplasia is not so rare as it was generally accepted. To reveal diffuse atypical hyperplasia of lung neuroendocri-ne cells, neuroendocrine markers should be used routinely in all resected specimens. In slices stained only with hematoxylin-eosin, without the use of neuroendocrine markers, this lesion may be practically overlooked in many cases. Probably we have missed some cases, because of inadequate sampling (only malignant tissue; or only a narrow rim of the pulmonary tissue, surrounding lung carcinoma, without included airways with respiratory epithelium). Therefore, we can conclude that diffuse idiopathic hyper-plasia of pulmonary neuroendocrine cells appears at least in 6% of random selected surgical specimens. It is more common in women, especially in the fifth and sixth decades.3 Atypical adenomatous hyperplasia is a quite common lesion. It may appear in 4-35% in different lung cancer resection specimens.3 Our surgical specimens were totally inadequate for the serious analysis of the incidence of atypical adenomatous hyperplasia. We have found it only in a few cases in the vicinity of adenocarcinoma. The localised lesions have usually less than 5 mm in diameter. Therefore, they are prac-tically inaccessible for usual bronchial and transbronchial biopsies and standard x-ray investigations. Aggressive small cell lung cancer has no known »ancestors« - precancerous lesions. Although the main goal of all these in-vestigations is detection of precancerous or early microinvasive cancer lesions, we sho-uld not exaggerate in overdiagnosing the actual lesions. It is much more reasonable to follow patients with suspicious lesions and to perform control biopsies. Conclusions Histology alone can find out early prein-vasive epithelial lesions, which may pro-ceed to invasive cancer types of non-small group. The additional molecular analysis with available immunohistology may be very helpful to find out the early signals of lung carcinogenesis. The patients with diffuse squamous metaplasia may develop reversible or even irreversible dysplastic changes. The follow-up of such patients and eventual rebiopsies are recommen-ded. The neuroendocrine lesions require a routine demonstration of neuroendocrine component with adequate marker. Radiol Oncol 2006; 40(Suppl 1): S67-S76. S76 Rott T et al / Pathohistology and detection of early lung cancer References 1. Rom WN, Hay JG, Lee TC, Jiang Y, Tchou-Wong KM. Molecular and genetic aspects of lung cancer. Amer J Resp Crit Care Med 2000; 161: 1355–67. 2. Rott T. Epidemiologija, etiopatogeneza in histološka klasifikacija pljučnih tumorjev. Med Razgl 2002; 41: 289–312. 3. World Health Organization classification of tumours. Pathology and genetics of tumours of the lung, pleura, thymus and heart. 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