Molecular predictive factors for progression of high grade preinvasive bronchial lesions.

Transcription

1 AJRCCM Articles in Press. Published on October 25, 2007 as doi: /rccm oc Molecular predictive factors for progression of high grade preinvasive bronchial lesions. Mathieu SALAÜN 1, Richard SESBOÜE 2, Sophie MORENO-SWIRC 3, Josette METAYER 3, Suzanna BOTA 1, Jeannette BOURGUIGNON 2 and Luc THIBERVILLE 1 1 : Clinique Pneumologique and Groupe LITIS - Quantif EA 4108, Rouen University Hospital, and Faculté de Médecine-Pharmacie, Rouen France 2 : INSERM U. 614, Faculté de Médecine-Pharmacie, Rouen, France. 3 : Service d Anatomie et Cytologie Pathologiques, and Laboratoire de génétique somatique des tumeurs, Rouen University Hospital, Rouen France. Corresponding author and request for reprint: Luc Thiberville, Clinique Pneumologique, Hôpital Charles Nicolle-CHU de Rouen, 1 rue de Germont, Rouen Cedex. Tel : , Fax : Luc.Thiberville@univ-rouen.fr Supported by a Research grant from the Comités Départementaux de la Ligue contre le Cancer (Eure et Seine Maritime), and the French Cancéropole Nord Ouest. Running title: Genetics of bronchial cancerization Subject category : 84 (Lung cancer : clinical aspects) Word count : 3878 Copyright (C) 2007 by the American Thoracic Society.

2 AT A GLANCE COMMENTARY Scientific knowledge on the subject: Several molecular alterations such as chromosome 3p deletions have been found in precancerous bronchial epithelium from high risk individuals. These factors had not been tested to predict the long term evolution of these lesions. What the study adds in the field : Chromosome 3p LOH is significantly associated with the progression of bronchial CIS on long term follow-up."

3 Abstract Rationale: The outcome of precancerous bronchial lesions is not well known, and their management is subject to controversy. Many molecular alterations are present in preinvasive lesions, but none has been assessed to predict the evolution of the lesions. Objective: To analyze the outcome of high grade precancerous lesions according to their molecular profile. Methods: 23 severe dysplasia and 31 carcinoma in-situ (CIS) in 37 patients were followed-up using repeated autofluorescence bronchoscopy over a 12 years period. Microdissection and PCR analysis were performed on paraffin tissue sections to assess loss of heterozygosity (LOH) and microsatellite instability on chromosome 3p, 5q, and 9p. Histology and molecular status at baseline were compared between 7 lesions that became invasive, 11 that relapsed after treatment, 17 that were eradicated with local treatment, and 19 that spontaneously regressed. Results: 94% of lesions that progressed or relapsed were CIS at baseline whereas 79% of spontaneously regressing lesions were severe dysplasia (p<0.0001). 3p and 9pLOH were more frequent in CIS than in severe dysplasia (p=0.03). In the whole group of lesions as well as in the CIS group, 3pLOH was strongly associated with progression (p< and p=0.02, respectively). Microsatellite instability was not associated with the outcome of the lesions. A therapeutic strategy based on the presence of 3p or 9p LOH, would have led to overtreat 6 lesions but would have missed only one among the 18 progressing lesions. Conclusions: Baseline histology and 3pLOH analysis appear to be useful to predict the outcome of high grade precancerous lesions. Word count for abstract : 250 words. 2

4 Keywords: Precancerous conditions; molecular biology; gene deletion; follow-up studies; bronchoscopy; disease progression. 3

5 Introduction The early detection of lung cancer appears to be crucial for the treatment efficacy and prognosis of this devastating disease (1). In the past decade, modern endoscopic methods of centrally located cancers such as autofluorescence endoscopy (2,3) have been developed that are able to detect and localize more effectively the early stages of epithelial bronchial cancerization, such as severe dysplasia and carcinoma in situ (CIS). However, because bronchoscopy is too invasive to be considered as a screening tool for bronchial cancers, a pre-screening method must be used in order to select the highest risk patients that could benefit from an endoscopic early detection program. A practical approach is to select the patients on the basis of clinical predictive factors such as occupational exposure to lung carcinogens, synchronous lung cancers and active smoking, which are also associated with a high frequency of proximal high grade lesions (4). Despite this prescreening selection, the efficacy of the early cancer endoscopic detection approach is currently limited by our restricted knowledge on the natural history of the preinvasive bronchial epithelium (5,6,7). Invasive squamous cell carcinoma of the bronchus is believed to develop from precancerous lesions beginning with hyperplasia, through the increasing degrees of dysplasia, and carcinoma in situ (CIS) (8). All the published series using autofluorescence bronchoscopy in individuals at high risk for lung cancer have found a very high frequency of preinvasive lesions, exceeding 50 % of the cases. (5,6,9,10). Therefore, a major issue is to more accurately differentiate lesions which are at high risk to progress into invasive cancers from the ones that are not (5,7). Using fluorescence endoscopy to follow-up the evolution of precancerous lesions, we have previously shown that the CIS lesions have the highest probability to persist, progress or relapse (5) as compared to severe dysplasia or lower grade lesions. 4

6 However, although the presence of a severe dysplasia or a CIS has been clearly associated with the occurence of a synchronous or a metachronous lung cancer (4,10,11), studies have currently failed to find reliable predictive factors of progression of high grade precancerous lesions at the individual level. Particularly, despite a greater probability for the highest grade lesions to progress, it currently appears impossible to make an accurate prediction of the long term evolution of a given lesion on the single basis of the histology results (5,6). With advances in molecular biology and the advent of micro-dissection technique, the genomic changes that are associated with the malignant bronchial transformation have been better characterized (12-17). The main molecular alterations found in invasive lung cancer consist in inactivating mutations of tumour suppressor genes, activating mutations of oncogenes, LOH, and amplifications of chromosomal regions (18). Recently, many of the abnormalities characteristic of the invasive tumors were also found in bronchial precancerous lesions (19) although with an overall lower frequency as compared with invasive cancers. Particularly, LOH of specific chromosomal markers such as in chromosome 3 and 9 were found to be very early event in bronchial cancerization, present as early as in reserve cell hyperplasia in both proximal and distal bronchi (12,19-21) In a previous article, we have evaluated chromosomal changes in a range of premalignant bronchial lesions of increasing severity, and found evidence of accumulation of gene losses with the progression of the lesions from one step to the next (12). We also performed a limited molecular follow-up study which found a good correlation between the persistance of the genomic abnormality over time and the evolution of the dysplastic lesion on follow-up (12). In this article, we are considering the hypothesis that the molecular abnormalities existing in high grade precancerous lesions such as severe dysplasia and CIS could be linked to their progression, and therefore could be used for their clinical management. The objective of this 5

7 study was to compare the molecular abnormalities present in CIS and severe dysplasia, between the progressing lesions and the regressing lesions. Patients and Methods Study design This study is part of the early lung cancer detection program which was initiated at Rouen University Hospital in In this program, individuals at high risk of developing lung cancer underwent a fluorescence bronchoscopy to detect early lung cancer and precancerous lesions in their bronchial tree. The evolution of preinvasive lesions found at baseline was assessed using repeated fluorescence bronchoscopy as described elsewhere (5). In this study, high grade lesions (SD and CIS) were followed-up for three months and CIS or severe dysplasia lesions that persisted at two consecutive endoscopies underwent endobronchial conservative treatment including cryotherapy, electrocaughtery or photodynamic therapy. The patients and the lesions (both locally treated and untreated) were followed up until death or the end point of the study (September 1 st, 2007) with a length of follow-up for the surviving patients of 115 months to 144 months. This made it possible to separate both patients and high grade lesions on the basis of their long term evolution. We used microdissection and PCR analysis of 3p, 5q and 9p microsatellites to assess LOH in high grade lesions, and correlated the results to the long term evolution of the lesions and the explored patients. Patients and lesions. 6

8 The inclusion criteria to enter the follow-up endoscopy program were a history of cigarette smoking of at least 30 pack-years and / or exposure to asbestos fibers and / or a history of ENT and / or lung cancer. All individuals were radiographically free of cancer at the time of inclusion. During the bronchoscopy, bronchial areas with suspected metaplasia, dysplasia or cancer under white light or under fluorescence examination were biopsied for pathologic examination. Fluorescence bronchoscopy and biopsy of the same sites were repeated over time according to the histopathology results found in the bronchi at baseline, and following a decision tree described elsewhere [5]. The selection criteria for the molecular study were: a baseline autofluorescent endoscopy showing the presence of at least one CIS or severe dysplasia, a clinical follow-up of a minimum of 9 years or until death, and sufficient archived material to achieve the molecular study. Thirtyseven patients who underwent the baseline autofluorescence endoscopy from February 1995 to October 1998 were selected for the molecular and follow-up study. From these patients 26 were part of the cohort explored in our previous work on short term evolution of precancerous bronchial lesions (5). Histological and molecular evaluation of bronchial biopsies Five 7-Mm-thick sections were cut from archived, formalin-fixed, paraffin-embedded blocks. Two slides were stained with hematoxylin-eosin (H&E) and were successively reviewed by two pathologists for diagnosis (SMS, JM). The lesions were classified according to the WHO 1999 criteria for preinvasive bronchial lesions (22). For this study we used a restrictive definition of high grade lesions including severe dysplasia and CIS. Lower grade lesions and microinvasive lesions were excluded from the molecular study. The three other cuts adjacent to the H&E stained material, were used for microdissection as described elsewhere (12,23). DNA was purified from microdissected cells ( cells) in 50 7

9 Ml of proteinase K buffer (Tris-HCl 50 mm, ph 8.8, EDTA 0.5 mm, Tween %, proteinase K 500 Mg/ml) for 48 h at 37 C, followed by heating at 100 C for 10 min. Reference genomic DNA was extracted for each subject from blood and extracted by standard techniques involving phenol-chloroform extraction, ethanol precipitation and solubilization in 50 Ml distilled water. Primers flanking polymorphic DNA motifs located at chromosomal regions 3p21-25, 5q21-22 and 9p22 were used to evaluate loss of heterozygosity (LOH) as described (12). PCR products on 3p, 5q were analyzed by electrophoresis in an 8% polyacrylamide gel followed by ethidium bromide or silver staining. LOH and microsatellite instability were studied on two 9p22 and two 3p21 markers in labelling the Reward primer 5 end with C6-FAM. 9p / 3p PCR products were analyzed using the automated ABI PRISM sequencer model 310 according to the supplier s protocol. Microsatellite instability was defined as the appearance of additional bands that differed in size compared to normal tissue DNA, according to established techniques (24). Definition of progression / stability / regression Changes in histology were regularly assessed to evaluate the progression / regression status of the lesions by comparison with the initial, baseline biopsy: «Regression» was defined as the spontaneous change from a non treated high grade lesion to a lower grade lesion (normal, reserve cell hyperplasia or regular metaplasia, mild or moderate dysplasia) without recurrence on follow-up; «Treatment Sensitive (or Stable) lesion» was defined as the persistence of a high grade lesion unchanged at three months, that was successfully treated without recurrence on follow-up. The progression group was divided in two subgroups : (a) «The treatment resistant lesions» were defined as the persistence of a high grade lesion unchanged, which justified an 8

10 endobronchial treatment and recurrence of this high grade lesion during follow-up without invasion of the basement membrane on long term follow-up, and (b) «progression to invasive lesions» was defined as the occurrence of an invasive cancer at the same bronchial area during follow-up. Inclusion period and follow-up. The high grade lesions considered in this study were diagnosed from February 1995 to October Patients were followed-up from the baseline endoscopy up to the end point of the study (September, 1 st, 2007) or death. Statistical analysis Comparisons were performed using Chi square tests or Fisher's exact test when required. A p value <0.05 was considered significant. RESULTS Regression / progression rate and patient s characteristics. 37 patients and 54 lesions (31 CIS, 23 SD) were included in the molecular study. The main characteristics of the patients and high grade lesions are summarized in table 1, where each patient is classified as CIS or SD according to the highest grade bronchial lesion found in their bronchial tree at baseline. The CIS and SD groups did not differ in age, gender, asbestos exposure, smoking status, history of ENT or lung cancer. According to our restrictive definition of regression / stability / progression, 19 lesions were classified as regressive and 17 lesions were considered as stable (also referred as treatment sensitive ), whereas 18 lesions 9

11 progressed on long term follow-up including 11 lesions that relapsed with the same histology after local treatment (also referred as treatment resistant ) and 7 lesions that became locally invasive. From these progressing lesions, 17, including all the lesions that progressed to an invasive cancer, were initially classified as CIS. Conversely, 7/17 of the stable lesions and 15 of the 19 spontaneously regressive lesions were classified as SD at baseline (p<0,0001, SD vs CIS). Long term follow-up. Details of long term follow-up for each lesion and patient, including molecular analysis, local evolution of the lesions, treatment administered, occurrence of an invasive cancer and its location, and the cause of death, are given in the online supplement (table 1E). A mean of 7 biopsies per lesion during follow-up (range 2-15) At the study Endpoint (sept 1st 2007), 10 patients were still alive without lung cancer with a follow-up of 115 mths to 144 mths and 2 patients were lost of follow-up without cancer at 102 and 120 mths. Twenty five patients died during follow-up, including 9 from lung cancer, from which 4 death could be directely attributable to the progression of the initial high grade lesion. Other deaths from cancer were related to 5 ENT cancer, one bladder cancer and one oesophageal cancer. Molecular abnormalities according to histology Table 2 summarizes the molecular abnormalities found in the bronchial baseline lesions according to histology. The number of lesions with at least one LOH was 13 and 26 in the severe dysplasia and CIS groups, respectively. The 5q deletion was less common in both CIS and SD groups (12/47). The frequency of 3p and 9p LOH was significantly higher in the CIS 10

12 group as compared to the SD group (p=0.01 and p=0.03, respectively). Microsatellite instability was observed in 10 CIS and 2 SD. 11

13 Progression / regression status and molecular abnormalities. Table 3 shows the molecular abnormalities according to the progression / regression rate in the entire group of lesions and the CIS group. In the entire group of lesions the existence of at least one LOH, and 3p LOH were strongly associated with progression of the considered lesion (p<0.01, and p< respectively). The association of 3 p deletion with 5q or 9p deletion was also associated with progression of the lesion. In the CIS group, 3p deletion and the occurrence of 3p or 5q deletion was significantly associated with progression (p<0,02 and p<0,04 respectively, Fisher s exact test). The microsatellite instability was not statistically associated with progression in the whole group or in the CIS group. Overall and Lung cancer specific survival curves. At the study endpoint, 14 patients had developed a lung cancer, from which 6 (7 lesions) were related to the progression of the initial CIS lesion, and 8 developed a cancer at a distant site from the initial high grade lesion. Table 4 details the outcome of the high grade lesions that were found in patients that developed an invasive cancer. Fig 1 shows the overall survival curve (median, 67 months) and the lung cancer specific survival curves (median not reached) for the whole group of patient. Survival curves of the patients according to the progression/regression status of the high grade lesions are displayed in figure 2. The median survival was not reached at the study end-point in the group of patient with lesion that regressed, whereas it was 65 months in the stabilisation (or treatment sensitive) group, 65 months in the treatment resistant group, and 44 months in the group of lesions that became invasive, respectively. 12

14 Figure 3 displays the overall survival according to the histology of the highest grade lesion, and to the presence or absence of 3p LOH in the highest grade lesion of a given patient. This figure shows a significant better survival in the 3p LOH free group (p=0.04) and a tendency for a longer survival in the SD group as compared with CIS groups. Influence of Lung or ENT cancer history on the high grade lesion outcome. 20 of the 37 patients included in the study had a previous history of lung or ENT cancer (table1e). The comparison of the lesions found in these patients and in individuals without previous malignancy did not find any difference in histology (SD versus CIS), 3p LOH frequency (p=1), or high grade lesion evolution on long term follow-up (p=0.63). DISCUSSION During the past decade, molecular studies of precancerous bronchial lesions have evidenced a progressive increase of 3p and 9p LOH frequency within bronchial preinvasive lesions with increasing severity (12, 19). This suggested that specific LOH or the association of several LOH could be used as markers of advanced bronchial disease, and thus could be tested as predictive factors of progression for a given lesion. In the present work, we postulated that the use of 3p, 5q and 9p LOH analysis may be helpful to predict the aggressiveness of bronchial severe dysplasia and CIS. In order to take into account the length of progression of the premalignant epithelium, this study required a long term, up to 12 years, follow-up of the patients using fluorescence bronchoscopy and repeated bronchial sampling over time. Whereas our series contains a large proportion of patients with a history of ENT or lung cancer, which are known to be at very high risk of developing a second cancer in the respiratory tract, we did not find any difference in the behaviour of the high grade lesions between the 13

15 patients with cancer history in their respiratory tract and the ones without. Therefore, it is likely that our results could be generalized to patients without previous malignancy. With a maximum of 144 months follow-up in 54 high grade bronchial lesions, we found that 3p LOH and histology (severe dysplasia versus CIS) were strong predictive factors of progression. We also found a tendency for the association of 3p and 9p LOH, or the association of 3p and 5q LOH with progression whereas there was no significant association between microsatellite instability and the lesion outcome. In this series, both prognostic values of histology and 3pLOH had a similar level of significance. However, 3pLOH was significantly associated with the progression when the CIS group was analyzed separately, and was constantly found in CIS that progressed into invasive cancer during follow-up. This demonstrated that the use of molecular analysis adds useful information over the use of histology alone for the prediction of progression. The results presented here are concordant with our previous findings on LOH frequency in precancerous bronchial lesions (12). Particularly, 3p LOH appears to be the more frequent alteration in high grade lesions, closely followed by 9p LOH, whereas 5q LOH appears to be a late event in bronchial carcinogenesis, more often associated with invasive lesions (12), but without prognosis value when found isolated from 3p or 9p deletion. On the other hand, 5q LOH may represent a molecular marker of clinical short term progression when associated with 3p LOH, indicating a fully transformed epithelium. The 3p/5q LOH allelotype was found in the present series in only 6 CIS, from which 2 progressed to invasive cancer at 4 months and 21 months follow-up, 3 were treatment resistant and only one was treatment sensitive. Close findings were observed in our previous molecular study where the only CIS displaying 3p, 5q and 9p alterations progressed into an invasive cancer despite endobronchial photodynamic therapy (12). The same observation was recently made in Foster s work, which followed up the 14

16 spontaneous evolution of two CIS in the same patient and found that the CIS that rapidly progressed into invasive lesion harboured the association of 5q, 3p and 9p LOH (25). To our knowledge, only three other studies on predictive molecular markers of preinvasive lesion progression have been published to date that all focused exclusively on preinvasive lesions of the oral cavity (26-28). These studies analysed 3p alterations in precancerous epithelium (3p LOH or FHIT gene alterations at 3p14), and all found a significant association between the molecular alteration and the progression from precursor oral lesions to oral squamous cell carcinoma (27, 28). These studies and ours confirm the major role of chromosome 3p alterations in the progression of the respiratory epithelium to invasive cancer, and its possible use as reliable predictive factor. The present study, which represents the first published work on the predictive value of molecular markers for high grade bronchial lesion outcome, may have important implications in the clinical practice. The few studies that have implemented a bronchoscopy follow-up procedure for the evaluation of precancerous lesion outcome have found a variable progression rate of the highest grade lesions (i.e. severe dysplasia and CIS), ranging from 100% and 87% of progression for CIS (5,6) to 37 % for severe dysplasia (5) and 17% for severe dysplasia or CIS (11). These differences appear mainly related to the inter-series differences regarding the definition of progression, the precocity of the treatment approaches of CIS and severe dysplasia (5, 29), the possible interference with systemic cancer treatment (11), as well as the follow-up duration of the patients. Therefore, there is still a debate on whether or not all high-grade preinvasive lesions are truly premalignant, and how they should be managed (30). In this context, the findings that 3p LOH frequency is linked to the long term progression / regression of the lesions appear to be valuable, in providing objective and reproducible elements that may help to decide whether a high grade bronchial lesion should be immediately 15

17 treated or not. In our series, only two spontaneously regressing severe dysplasia and none of the 4 long term spontaneously regressing CIS harboured a 3p LOH at baseline. Therefore, a 3p LOH based treatment decision approach would have led to only two "overtreated" lesions. On the other hand, a treatment approach based on the presence of 3p or 9p LOH, would have resulted in the treatment of 6 lesions that would have spontaneously regressed but would have missed only one among the 18 progressing lesions. We believe that this study also lends strong support to the WHO classification for premalignant bronchial lesions that differentiates bronchial CIS from severe dysplasia, in confirming their different outcomes, as we previously observed (5), but also in providing evidences of differences at the molecular level. In the present work, after a classification of the high grade lesions based on a careful, double histology lecture, we found a significantly lower proportion of long term regression in CIS as compared with severe dysplasia and a trend for a shorter survival in patients presenting with CIS. We also demonstrated the clinical value of both the molecular analysis and the regression status, as the patients harbouring a high grade bronchial lesion that regressed and those without 3pLOH appear to have the longer survival. Finally, we clearly showed that the molecular abnormalities existing in severe dysplasia (i.e. 3p and 9 p LOH) are significantly less frequent as compared to CIS. The distinction between severe dysplasia and CIS was not always recognized in the literature as clinically relevant (11), presumably because of the difficulties encountered for the histological classification of high grade lesions, leading to inter-observational variation in the histology interpretation (11). The significant differences in 3p and 9p LOH frequency between CIS and severe dysplasia observed in our study and their association with different outcomes suggest that the molecular analysis of the highest grade lesions could also help in their histoprognostic classification. 16

18 The study presented here has several limitations. It only considers 4 molecular events, encompassing a relatively small number of chromosomal loci which correspond to putative tumour suppressor genes that are not yet identified. In particular, it is recognized that several LOH hotspots exist on the short arm of chromosome 3 in lung cancer and premalignant lesions (31). Each of these specific hotspots and / or the size of the 3p allele loss regions (31) may not have the same predictive value in terms of lesion aggressiveness. However, a more extensive study of 3p LOH would have been impossible in our series in addition to 9p and 5q analysis, because of the small size of the material that is available from paraffin embedded archived samples. Another possible limitation is related to our treatment decision tree, which includes an endobronchial conservative treatment of the high grade lesions that persisted unchanged at three months, which obviously interferes with the natural evolution of the bronchial disease. To overcome this known limitation, we used in this study a very restrictive definition of progression, including local progression to invasive cancer and local recurrence after the endobronchial treatment of the lesion on follow-up. This made it possible to accurately classify the more aggressive lesions in the progression group. We also selected in our database the lesions for which we had a very long term follow-up up to 12 years, which ascertains that the not treated regressing lesions were truly benign ones. Besides the length of follow-up, the strength of our study also relies on the robustness and the simplicity of the molecular methods that have been used, which could be easily implemented in the clinical practice. The microdissection of paraffin embedded tissue section using LASER capture is currently a simple and reliable technique that is routinely used in our somatic genetic tumour laboratory. In this series, the microdissection of tissue paraffin embedded sections appeared mandatory for several reasons: mainly the very small size of the sampled preinvasive epithelium (frequently a few hundreds of cells), the need to avoid 17

19 contamination from normal or lower grade adjacent precancerous epithelium, as well as the need to analyze the very precise diagnostic epithelial area. Coupled with PCR analysis, the method is inexpensive and the results can be available in less than a week s time, which assures that they could be effectively integrated in a treatment decision tree. In the future, more sophisticated methods such as quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF) may replace the single PCR method, in allowing simultaneous amplification under quantitative conditions of multiple dye-labeled targets from both tumor and nonmalignant tissues (32). The QMPSF technique, which also appears very robust and rapidly performed, has been successfully used in our laboratory to better characterize quantitative genetic alterations in colorectal cancers (32). However, it needs to be adapted to very small fixed and paraffin embedded samples, which was not possible at the time of this work. A QMCSF prospective study on fresh or frozen tissue could however be performed in the future. Taken together, these data indicate that molecular analysis can be usefully added to the histopathology grading of precancerous lesions in order to better classify the lesions and identify the more aggressive ones. They also suggest the possible use of a simple, molecularbased management decision tree of high grade precancerous lesions. In this decision tree, the high grade lesions that harbour a 3p LOH would be the more aggressively treated, since their probability of progression appears very high (76 % in our series), whereas the lesions without 3p or 9p molecular alterations, whose probability of progression is only 5 %, would be submitted to a follow up procedure. In the future, lower grade lesions such as mild and moderate dysplasia could also be assessed using an equivalent molecular approach. In a previous work, we demonstrated the low probability of progression of low grade precancerous bronchial lesions to high grade at 24 months follow-up, as well as the association of their progression with a field of cancerization effect (12). Based on this observation, our group is currently conducting a prospective, multicentric, 3 year bronchoscopic follow-up study of low 18

20 grade precancerous lesions (clinical trials.gov ID : NCT ) (33). The results of this trial involving 350 high risk individuals will be available in 2009, and will help to assess the probability of progression of the lowest grade lesions as well as their relationship with the molecular alterations. Future studies will also analyze the cost effectiveness of an approach integrating the molecular analysis into the management decision tree of high grade preinvasive bronchial lesions. 19

21 References 1 : Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer Statistics, CA Cancer J Clin 2007;57: : Lam S, Kennedy T, Unger M, Miller YE, Gelmont D, Rusch V, Gipe B, Howard D, LeRiche JC, Coldman A, Gazdar AF. Localization of bronchial intraepithelial neoplastic lesions by fluorescence bronchoscopy. Chest 1998;113: : Kennedy TC, Hirsch FR, Miller YE, Prindiville S, Murphy JR, Dempsey E, Proudfoot S, Bunn PA, Franklin WA. A randomized study of fluorescence bronchoscopy versus white-light bronchoscopy for early detection of lung cancer in high risk patients. Lung Cancer 2000;29:A : Paris C, Benichou J, Bota S, Sagnier S, Métayer J, Eloy S, Auliac JB, Nouvet G, Thiberville L. Occupational and nonoccupational factors associated with high grade bronchial pre-invasive lesions. Eur Respir J 2003;21: : Bota S, Auliac JB, Paris C, Métayer J, Sesboüé R, Nouvet G, Thiberville L. Follow-up of bronchial precancerous lesions and carcinoma in situ using fluorescence endoscopy. Am J Respir Crit Care Med 2001;164: : Breuer RH, Pasic A, Smit EF, Van Vliet E, Vonk Noordegraaf A, Risse EJ, Postmus PE, Sutedja TG. The natural course of preneoplastic lesions in bronchial epithelium. Clin Cancer Res 2005;11:

22 7 : Auer G, Ono J, Nasiell M, Caspersoon T, Kato H, Konaka C, Hayata Y. Reversibility of bronchial cell atypia. Cancer Res 1982;42: : Auerbach O, Stout AP, Hammond EC, Garfinkel L. Changes in bronchial epithelium in relation to cigarette smoking and cancer of the lung. N Engl J Med 1961;265: : Lam S, leriche JC, Zheng Y, Coldman A, MacAulay C, Hawk E, Kelloff G, Gazdar AF. Sex-related differences in bronchial epithelial changes associated with tobacco smoking. J Natl Cancer Inst 1999;91: : Loewen G, Natarajan N, Tan D, Nava E, Klippenstein D, Mahoney M, Cummings M, and Reid M. Autofluorescence bronchoscopy for lung cancer surveillance based on risk assessment. Thorax 2007;62: : George PJ, Banerjee AK, Read CA, O Sullivan C, Falzon M, Pezzella F, Nicholson AG, Shaw P, Laurent G, Rabbitts PH. Surveillance for the detection of early lung cancer in patients with bronchial dysplasia. Thorax 2007;62: : Thiberville L, Payne P, Vielkinds J, LeRiche J, Horsman D, Nouvet G, Palcic B, Lam S. Evidence of cumulative gene losses with progression of premalignant epithelial lesions to carcinoma of the bronchus. Cancer Res 1995;55:

23 13 : Brambilla E, Gazzeri S, Lantuejoul S, Coll JL, Moro D, Negoescu A, Brambilla C. p53 mutant immunophenotype and deregulation of p53 transcription pathway (Bcl2, Bax, and Waf1) in precursor bronchial lesions of lung cancer. Clin Cancer Res 1998;4(7): : Brambilla E, Gazzeri S, Moro D, Lantuejoul S, Veyrenc S, Brambilla C. Alterations of Rb pathway (Rb-p16INK4-Cyclin D1) in preinvasive bronchial lesions. Clin Cancer Res 1999;5: : Westra WH, Slebos RJ, Offerhaus GJ, Goodman SN, Evers SG, Kensler TW, Askin FB, Rodenhuis S, Hruban RH. K-ras oncogene activation in lung adenocarcinomas from former smokers. Cancer 1993;72(2): : Okamoto A, Hussain SP, Hagiwara K, Spillare EA, Rusin MR, Demetrick DJ, Serrano M, Hannon GJ, Shiseki M, Zariwala M, Xiong Y, Beach DH, Yokota J, Harris CC. Mutations in the p16ink4/mts1/cdkn2, p15ink4b/mts2, and p18 genes in primary and metastatic lung cancer. Cancer Res 1995;55: : Gazzeri S, Brambilla E, Caron de Fromentel C, Gouyer V, Moro D, Perron P, Berger F, Brambilla C. p53 genetic abnormalities and myc activation in human lung carcinoma. Int J Cancer 1994;58: : Rom WN, Hay JG, Lee TC, Jiang Y, Tchou-Wong KM. Molecular and genetic aspects of lung cancer. Am J Respir Crit Care Med 2000;161:

24 19 : Wistuba II, Behrens C, Milchgrub S, Bryant D, Hung J, Minna JD, Gazdar AF. Sequential molecular abnormalities are involved in the multistage development of squamous cell carcinoma. Oncogene 1999;18: : Sundaresan V, Ganly P, Hasleton P, Rudd R, Sinha G, Bleehen NM, Rabbits P. p53 and chromosome 3 abnormalities, characteristics of malignant lung tumours, are detectable in preinvasive lesions of the bronchus. Oncogene 1992;7: : Hung JY, Kishimoto Y, Sugio K, Virmani A, McIntire DD, Minna JD, Gazdar AF. J A M A 1995;273: : Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Histological typing of lung and pleural tumours with contributions by pathologists from 14 countries. In: World Health Organization international histological classification of tumors, XIII, 3 rd ed. Berlin/Heindelberg:Springer-Verlag; : Schutze K, Posl H, Lahr G. Laser micromanipulation systems as universal tools in cellular and molecular biology and in medicine. Cell Mol Biol 1998;44: Ninomiya H, Nomura K, Satoh Y, Okumura S, Nakagawa K, Fujiwara M, Tsuchiya E and Ishikawa Y. Genetic instability in lung cancer: concurrent analysis of chromosomal, mini- and microsatellite instability and loss of heterozygosity. British Journal of Cancer 2006; 94:

25 25 : Foster NA, Banerjee AK, Xian J, Roberts I, Pezella F, Coleman N, Nicholson AG, Goldstraw P, George JP, Rabbitts PH. Somatic changes accompanying lung tumor development. Genes Chromosomes Cancer 2005;44: : Mao L, Lee JS, Fan YH, Ro JY, Batsakis JG, Lippman S, Hittleman W, Hong WK. Frequent microsatellite alterations at chromosome 9p21 and 3p14 in oral premalignant lesions and their value in cancer risk assessment. Nat Med 1996;2: : Rosin MP, Cheng X, Poh C, Lam WL, Huang Y, Lovas J, Berean K, Epstein JB, Priddy R, Le ND, Zhang L. Use of allelic loss to predict malignant risk for low-grade oral epithelial dysplasia. Clin Cancer Research 2000;6: : Kujan O, Oliver R, Roz L, Sozzi G, Ribeiro N, Woodwards R, Thakker N, Sloan P. Fragile histidine triad expression in oral squamous cell carcinoma and precursor lesions. Clin Cancer Res 2006;12: : Venmans BJ, Van Boxem TJ, Smit EF, Postmus PE, Sutedja TG. Outcome of bronchial carcinoma in situ. Chest 2000;117: : Banerjee AK, Pamela H. Rabbitts, P. Jeremy M. George, Thiberville L, Bota S, Auliac JB, Paris C, Métayer J, Nouvet G, Sesboüé R. Are all high-grade preinvasive lesions premalignant, and should they all be treated? Am J Respir Crit Care Med 2002;165: : Wistuba II, Behrens C, Virmani AK, Mele G, Milchgrub S, Girard L, Fondon III JW, Garner HR, McKay B, Latif F, Lerman MI, Lam S, Gazdar AF, Minna JD. High resolution 24

26 chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res 2000;60: : Killian A, Di Fiore F, Le Pessot F, Blanchard F, Lamy A, Raux G, Flaman JM, Paillot B, Michel P, Sabourin JC, Tuech JJ, Michot F, Kerckaert JP, Sesboüé R, Frebourg T. A simple method for the routine detection of somatic quantitative genetic alterations in colorectal cancer. Gastroenterology 2007;132: : Reference study available from 25

27 Table legends. Table 1: Patient characteristics Legend : $ Chi square, * Fisher s exact test, T test (Student) Table 2: Molecular alterations according to histology Legend : $ Chi square test, *Fisher s exact test, Inv. = lesion that became invasive, Tt R = treatment resistant, TtS = treatment sensitive, Table 3: Evolution of the lesions according to allelic loss and histology. Legend : $ Chi square test, *Fisher s exact test, Tt R = treatment resistant, TtS= treatment sensitive Table 4 : Patients that developped an invasive lung cancer. Legend : * : Last biopsy at 10 mths Follow-up. Abbreviations : RCH : Reserve cell hyperplasia, SD : Severe Dysplasia ; CIS : Carcinoma in-situ. Y : LOH present ; N : No LOH ; - : Uninformative SCC : squamous cell lung cancer, SCLC : small cell lung cancer. 26

28 Figure legends Figure 1: Overall survival curve, lung cancer specific survival curve (all patients) and survival of patients that developped an invasive lung cancer. 1 : Overall survival (median : 67 months) 2 lung cancer specific survival curve (median :not reached) 3: Patients that developped an invasive cancer (median : 22 months) Figure 2: Survival according to evolution of the highest grade lesion. Figure 2A: 1: Progression to Invasive (median: 44 months) 2: Treatment resistant (median: 65 months) 3: Treatment sensitive (median: 47 months) 4: Spontaneous Regression (median: not reached) Figure 2B: Spontaneous regression versus other lesions 1: Progression to Invasive, treatment resistant and treatment sensitive lesions (Median : 65 months) 2: Spontaneous Regression (median: not reached) p=0.08 (Log Rank) Figure 3: Survival according to baseline 3p LOH status (3A) and histology of the highest grade lesion (3B) Figure 3A: Survival according to 3p LOH 1: 3p LOH (median survival: 37 month) 2: Absence of 3p LO p=0,04 (Log-Rank) Figure 3B: B: Survival according to baseline histology 1 : CIS (median survival : 44 months) 2 : Severe dysplasia (median survival : 77 months) p=0,15 (Log-Rank) 27

29 Tables Table 1 Patient characteristics Patients with SD Patients with CIS p value as highest grade lesion (n = 13) as highest grade lesion (n = 24) Age at baseline (years) 59 (40-76) 60 (46-74) 0.73 Mean (range) Male / female 12/ 1 24 / * Smoking (P.Y) 48 (+/-17) 45 (+/-15) 0.68 Mean (SD) Asbestos exposure * ENT cancer history * Lung cancer history $ $ Chi square * Fisher s exact test T test (Student) 28

30 Table 2 Molecular alterations according to histology SD (n = 23) CIS (n = 31) p At least one LOH / lesions 13/23 26/ $ LOH 3p / informative lesions LOH 5q / informative lesions LOH 9p / informative lesions Microsatellite instability / Analyzed lesions 3p and 9p LOH / informative lesion 3p or 9p LOH / informative lesion 3p and 5q LOH / informative lesion 3p or 5q LOH / informative lesion 5/22 16/ $ 5/22 7/ /17 20/ $ 2/17 10/ * 1/16 10/ * 10/23 26/ $ 1/21 5/ * 9/23 18/ Inv./Tt R./Tt S./Regression. 0/1/7/15 7/10/10/4 <0.0001* $ Chi square test, *Fisher s exact test, Inv. = lesion that became invasive, Tt R = treatment resistant, TtS = treatment sensitive, 29

31 Table 3: Evolution of the lesions according to allelic loss and histology. High grade precancerous lesions (CIS and SD) CIS Invasive Tt R Tt S Regression p Invasive Tt R Tt S Regression p Lesions with at least one LOH 7/7 10/11 13/17 9/ $ 7/7 9/10 8/10 2/ p LOH 6/6 7/11 6/16 2/17 <0.0001* 6/6 6/10 4/9 0/ $ 5q LOH 3/5 3/10 1/15 5/ * 3/5 3/9 0/8 1/ p LOH 5/7 6/9 10/15 5/ * 5/7 6/9 7/10 2/3 1 Microsatellite instability 3p and 9p LOH 2/7 4/11 5/15 1/ * 2/7 4/10 4/10 0/ /6 3/9 3/14 1/ * 4/6 3/9 3/9 0/ p or 9p LOH 7/7 10/11 13/17 6/19 <0.0001* 7/7 9/10 8/10 2/ p and 5q LOH 3p or 5q LOH 2/4 3/10 0/15 1/ * 2/4 3/9 0/8 0/ /7 7/11 7/16 6/ * 7/7 6/10 4/9 ¼ 0.04 $ $ Chi square test, *Fisher s exact test, Tt R = treatment resistant, TtS= treatment sensitive 30

32 Table 4 Patients Histology grade and location of the initial lesion Evolution of the initial lesion location of invasive tumor (histology) Time between invasive cancer diagnosis and first bronchoscopy (months) Grade of the initial lesion when cancer developped Molecular analysis 3p LOH / 5q / 9p / MI 3 RB6a (CIS) RB4a (SD) Invasive Regression RB6a (SCC) 21 Invasive Unavailable* Y/Y/Y/N N/Y/N/N 5 LB9 (CIS) LMB (CIS) Invasive Treatment sensitive LB9 (SCC) 31 Invasive SD Y/-/N/N Y/-/N/N 6 RB1 (CIS) Regression Mediastinum (SCC) 42 Moderate Dysplasia N/N/-/N 8 LB6 (CIS) Invasive RB1 (SD) Regression 12 LB9 (SD) Regression LB6 (SCC) 4 Troncus intermedius (SCLC) 45 Invasive Normal Mild dysplasia Y/Y/N/N N/-/N/N N/-/N/N 15 RB3 (CIS) Invasive RUL (SCC) 42 Invasive N/N/Y/N 17 MC (SD) Treatment sensitive LB6 (SCC) 3 MD N/N/Y/N 23 RUL (CIS) Treatment sensitive LUL (SCC) 19 Inflammarory N/N/Y/N 24 LB4 (CIS) Treatment resistant LLL (Adenocarcinoma) LB6 (CIS) Treatment sensitive Trachea (SCC) 3 27 Trachea (CIS) Trachea (CIS) Invasive Invasive Trachea (SCC) 3 Reserve Cell Hyperplasia Reserve Cell Hyperplasia Invasive Invasive Y/Y/Y/N -/N/Y/N Y/N/Y/Y Y/N/N/Y 30 RB6 (CIS) Treatment resistant LUL (SCC) 8 CIS N/Y/N/N 33 RB2 (CIS) LB6 (CIS) Invasive Regression RB2 (SCC) 49 Invasive Normal -/Y/Y/N -/Y/Y/N 36 LMB (CIS) Treatment resistant RUL (SCC) 12 Moderate Dysplasia Y/-/Y/Y

33 Figures Figure 1

34 Figure 2: Survival according to evolution (highest grade lesion) Figure 2A: Figure 2B: Spontaneous regression versus other lesions 1

35 Figure 3: Survival according to baseline 3p LOH status and histology of the highest grade lesion Figure 3A: Survival according to 3p LOH (p<0.04, Log Rank) Figure 3B: B: Survival according to baseline histology (p=0.15, Log Rank) 2

36 On line Supplement : Molecular predictive factors for progression of high grade preinvasive bronchial lesions. Mathieu SALAÜN, Richard SESBOÜE, Sophie MORENO-SWIRC, Josette METAYER, Suzanna BOTA, Jeannette BOURGUIGNON and Luc THIBERVILLE

37 Results Table 1E : Details of patients and lesions outcome. Molecular analysis, local treatment, number of biopsies per site, delay between first and last biopsy, time to progression, last histology and patients outcome.

38 Patients Baseline histology* Molecular analysis 3p/5q/9p/ MI Local treatment (nb) Evolution Time to Progression to invasive lesions (mths) Number of biopsies Last biopsy histology (II) Time between first and last biopsy Location of High grade lesion Location of invasive tumor Patient s follow-up (cause of death) 1 e CIS N/N/-/- 0 R 10 N 89 RB e SD Y/N/N/Y C (1) Ts 8 md 65 Trachea - 69 (Sepsis) 3 SD N/Y/N/N (0) R 2 RCH 10 RB4 RB6 49 (cardiac CIS Y/Y/Y/N C (4) I SCC 48 RB6a RB6 failure) 4 e SD N/N/N/N (0) R 5 N 25 RB2-78 (ENT Cancer) 5 e CIS Y/-/Y/N PDT (6) I SCC 44 LB9 LB9 55 (Lung CIS Y/-/N/N PDT (1) Ts 3 SD 26 LMB LB9 cancer) 6 e CIS N/-/N/N (0) R 8 MD 50 RB1 Mediastinum 68 (Lung cancer) 7 e SD N/N/Y/N C (1) Ts 8 MD 56 Trachea - 66 (ENT Cancer) 8 CIS Y/Y/N/N PDT (1) I 4 8 CIS 11 a LB6 LB e CIS Y/N/Y/N PDT (1) Ts 4 N 6 b RB6-120 (Lost of follow-up) CIS N/N/Y/N (0) R 10 N 103 RB6a 10 SD Y/N/-/- PDT (1) Ts 12 N 112 LB CIS N/N/N/N PDT (1) Ts 11 N 112 LB6 11 SD N/N/N/N E (1) Ts 8 MD 130 RB6 - SD N/N/Y/N (0) R 7 N 128 LB SD N/N/-/- (0) R 4 N 21 RB1 Troncus 58 (Lung SD N/N/-/- (0) R 5 md 34 LB9 intermedius cancer) 13 e SD N/N/N/N (0) R 10 N 95 RUB CIS Y/Y/Y/N PDT (1) Ts 5 N 21 LB3-23 (ENT cancer) 15 CIS Y/-/Y/N PDT (3) I SCC 47 RB3 RUL 77 (Lung cancer) SD N/N/N/Y (0) R 3 MD 4 RB9 16 e CIS N/N/Y/Y PDT (1) Ts 3 RCH 12 UDB - 14 (Acute pneumonia)

39 Patients Baseline histology* Molecular analysis 3p/5q/9p/ MI Local treatment (nb) Evolution Time to Progressi on to invasive Number of biopsies Last biopsy histology II Time between first and last biopsy Location of High grade lesion Location of invasive tumor Patient s follow-up (cause of death) 17 e SD N/N/Y/N PDT (1) Ts 3 MD 6 Carena LB6 17 (Schock) 18 e SD -/N/Y/N (0) R 4 N 36 RB CIS N/N/Y/Y C (3) TR 15 SD 55 RB7 CIS N/N/N/Y C (3) TR 14 MD 50 RML 19 e CIS N/N/N/Y C(1) Ts 10 MD 42 RUL SD Y/N/-/N PDT (2) TR 12 SD 52 RB e SD Y/Y/-/- (0) R 4 N 16 LB SD N/N/N/N (0) R 9 M 91 RB1-3 SD N/N/N/N (0) R 10 RCH 76 RB CIS Y/N/Y/Y PDT (1) Ts 4 RCH 11 RB5a - 23 CIS N/N/Y/N CT Ts 2 N 10 RUB RB6 24 e CIS Y/Y/Y/N PDT (2) TR 3 RCH 19 LB4 LLL CIS Y/N/N/N PDT (2) TR 5 CIS 11 UDB 25 e CIS Y/N/Y/N PDT (2) TR 3 RCH 6 LB3 26 e CIS -/-/Y/N PDT (1) Ts 3 RCH 3 C LB6 Trachea 27 e CIS Y/N/N/Y C (1) I 7 3 SCC 9 Trachea CIS Y/N/Y/Y C (1) I 7 3 SCC 9 Trachea - 66 (Bladder cancer) 34 (ENT Cancer) Trachea 38 (Oesophagal cancer) 27 (Lung cancer) 21 (Respiratory failure) 17 (ENT Cancer) 9 (Lung cancer) 12 (Lung cancer) 28 SD N/Y/N/N (0) R 7 md 63 LB1-2 SD N/Y/N/N (0) R 6 N 63 LB3-131

40 Patients Baseline histology* Molecular analysis 3p/5q/9p/ MI Local treatment (nb) Evolution Time to Progressi on to invasive Number of biopsies Last biopsy histology II Time between first and last biopsy Location of High grade lesion 29 SD N/N/-/- PDT (1) Ts 6 MD 37 RB9 - Location of invasive tumor 30 e CIS N/N/Y/N CT (1) TR 3 CIS 10 RB6 LUL Patient s follow-up (cause of death) 51 (Myocardial infarction) 31 CIS N/N/Y/Y PDT (1) Ts 8 md 55 LB (Lung Cancer) 32 SD N/N/N/N (0) R 3 MD 13 RB8a SD N/Y/Y/N (E) Ts 3 MD 13 LB (Lost of follow-up) d CIS -/Y/Y/N PDT(2) I 11 6 SCC 54 RB2 33 e CIS -/Y/Y/N (0) R 4 N 49 LB6 RUL 84 (Lung cancer) 34 CIS Y/N/Y/Y PDT(1) ; E(1) TR 5 N 87 LB8-92 (Respiratory failure) SD Y/-/Y/N (0) R 10 N 115 RB8a 35 e CIS N/-/Y/N 36 CIS Y/Y/N/N PDT(2) E(1) CT(1) PDT (2)CT;RT TR 11 N 112 LMB TR 5 md 12 Trachea RUL 37 CIS Y/Y/-/Y E (2) TR 9 CIS 44 LB (Heart failure) 75 (Acute pancreatitis)

41 Abbreviations : *: CIS = carcinoma in situ ; SD = Severe dysplasia : MI = Microsatellite Instability; Y = existing loss of heterozygisity or microsatellite instability ; N = no LOH or MI : 0 = No treatment ; C = Cryotherapy ; PDT = PhotoDynamic Therapy ; E : Electrocaughtery ; RT : Radiotherapy ; CT : Chemotherapy ; ct : endobronchial curietherapy : R : Regression ; Ts : Treatment sensitive ; TR : Treatment resistant ; I : Invasive (II) : N : Normal ; RCH : Reserve Cell Hyperplasia ; M : Metaplasia ; md : Mild Dysplasia ; MD : Moderate Dysplasia ; SD : Severe Dysplasia ; CIS : Carcinoma in situ ; SCC : Squamous Cell Carcinoma, SCLC : Small Cell Lung Cancer. Comments : a : LB6 : The CIS progressed to invasive, was therefore treated locally but relapsed with CIS histology. Subsequent treatment consited of a left lower lobectomy. b : RB6 : This CIS was treated by one course of PDT. The control biopsy showed a complete response at 6 months. The patient refused further endoscopic follow-up, but is alive and well at 120 months without any sign of tumor recurrence. Lesion is considered as long term treatment sensitive. c : This patient developped an invasive cancer in the upper part of the trachea, therefore, the LB6 lesion was not sampled after the 3rd months following treatment. d : patient 32 refused bronchial follow-up but was alive and well without sign of lung cancer after 102 months e : Patients with previous history of lung or ENT cancer.