3p Microsatellite Alterations in Exhaled Breath Condensate from Patients with Non–Small Cell Lung Cancer

1

3p microsatellite alterations in exhaled breath condensate from non-

small cell lung cancer patients

Carpagnano, Giovanna GE, Foschino-Barbaro, Maria Pia MP, *Mule’, Giuseppina,

**Resta, Onofrio, ***Tommasi, Stefania,***Mangia A, ****Carpagnano, Francesco,

*Stea, Gaetano, *Susca, Antonella, **Di Gioia, Giusseppe, ***De Lena, Mario,

***Paradiso, Angelo

Institute of Respiratory Disease, University of Foggia, Italy; *ISPA, CNR, Bari, Italy;**Institute of

Respiratory Disease, University of Bari, Italy; ***Clinical Experimental Oncology Lab National

Cancer Institute, Bari; ****Department of Thoracic Surgery, San Paolo Hospital, Bari, Italy.

Address for correspondence: Giovanna Elisiana Carpagnano Via De Nicolo’ 5, 70121, Bari Italy Tel 00390805301321 Fax 00390805045362 [email protected] Short running head: Microsatellite alterations in NSCLC Partially supported by Special Project "Programma Italia-USA – Farmacogenomica Oncologica" 2004. Category number: 85

Introduction: 531

Methods: 1017

Results: 757

Discussion: 1204

AJRCCM Articles in Press. Published on June 9, 2005 as doi:10.1164/rccm.200503-439OC

Copyright (C) 2005 by the American Thoracic Society.

Abstract

Rationale: The still high mortality for lung cancer urgently requires the availability of new non-

invasive diagnostic tools destined to more effective early diagnosis and screening programs.

Recently, exhaled breath condensate (EBC) has been proposed as a useful tool to obtain biological

information on lung cancer disease.

Objectives: The present study checked for the feasibility of microsatellite alteration (MA) analysis in

the EBC-DNA from NSCLC patients with respect to alterations found in whole blood (WB) DNA.

Methods: Thirty patients with a histological evidence of NSCLC and 20 healthy subjects were

enrolled. All subjects had allelotyping analysis on DNA from EBC and WB-DNA of a panel of

microsatellites (D3S2338, D3S1266, D3S1300, D3S1304, D3S1289) located in chromosomal region

3p. Results from healthy and cancer subjects, and from EBC and WB were compared. Furthermore, the

relationships with smoking habit and clinical-pathological tumour features were considered.

Measurements and Main Results:

MA were found in 53% of EBC-DNA and in 10% of WB-DNA loci investigated from NSCLC patients

(p< 10-6); conversely, MA were present only in 13% in EBC-DNA and 2% in WB-DNAs informative

loci of healthy subjects. In NSCLC patients a direct association between number of MA detected in

EBC-DNA and tobacco consumption was observed.

Conclusions: In the present paper, for the first time we provide evidence that EBC-DNA is highly

sensitive in detecting MA from NSCLC and healthy subjects. Furthermore, MA information seem to be

directly related with tobacco consumption and then, potentially applicable to screening and early

diagnosis programs for NSCLC patients.

Key words: DNA, LOH, chromosome 3, NSCLC susceptibility

Word count: 249

3

Lung cancer remains the most frequent tumour and cause of cancer death in men and

recently its frequency is significantly increasing also in female gender (1). Although a large

number of potential causes for lung cancerogenesis have been hypothesized, an important role has

been universally reported for tobacco use accounting for more than 80% of lung cancer

development (2).

Despite the big efforts to find new more sensitive diagnostic tools able to anticipate the

diagnosis of this cancer, a significant percentage of patients will never undergo surgery because of

a too extended disease at diagnosis. To try to anticipate the diagnosis, also the possibility to

conduct screening programs in asymptomatic high-risk population groups have been considered; to

this purpose, cytology of the sputum, circulating tumour biomarkers, chest-X ray, MNR, etc have

been attempted sometimes with interesting but still inconclusive results (3).

A completely different approach concerns the possibility to look for bio-molecular markers

of lung cancerogenesis or tumor progression potentially permitting the individualization of early

cell damages and a non invasive staging of the disease. In fact, nowadays it is generally accepted

that lung cancer results from the occurrence of a number of genetic alterations in oncogenes and

tumour suppressor genes (4-6) that are potential markers either for screening procedures or for

earlier detection in patients with non small-cell lung cancer (NSCLC) (5,7,8). Several DNA

alterations taking place during the development of cancer (gene mutation, microsatellite instability,

promoter methylation and overexpression) have been already identified in different biological

samples of patients with lung cancer (9). However, tissues utilised for molecular studies turn out to

be difficult to harvest as they require highly invasive techniques that make them poorly suitable for

wider screening.

Recently, Gessner have demonstrated the possibility to detect human DNA in the exhaled

breath condensate, EBC (8). The EBC is a fluid coming from the airways which can be collected

by means of a very easy, completely non-invasive, repeatable procedure that is also well accepted

4

by patients (10-15) For this reason, the analysis of genetic characteristics in EBC could really

represent the way for the non invasive identification of early markers of NCSLC.

Recent insights into the molecular basis of cancer have recognized the occurrence of some

triggering molecular events likely to result in the development of lung cancer including the

microsatellite instability (MI) and loss of heterozygosity (LOH) (16,17). Microsatellites are

repetitive nucleotide sequences of varying lengths, which are scattered throughout the genome,

between and within genes. They have been used as markers for genetic mapping because they are

highly polymorphic and stably inherited (18-20) Previous studies have shown the presence of

microsatellite alterations (MA) in NSCLC with a variable frequency depending on the number and

loci studied and on the clinical-pathological characteristics of the patients (21,22). Therefore, MA

has been recently proposed as an early markers also in lung carcinogenesis (23)(24).

The aim of the present study has been to check for the feasibility of MA analysis in the

EBC-DNA from NSCLC patients. Furthermore, differences between results from DNA of healthy

and cancer subjects, and from EBC-DNA and of whole blood DNA (WB-DNA) were compared.

Finally, the relationships with smoking habit and clinical-pathological tumour features were

considered.

5

Methods

Characteristics of Patients

The study population consisted of 30 patients (19 men, mean age±SD: 63±8 years) who

achieved a histological diagnosis of NSCLC at the Department of Thoracic Surgery, San Paolo

Hospital, Bari and at Department of Respiratory Disease of Foggia University. Twenty healthy

controls, negative for cancer chest-scan, have been also enrolled (13 men, mean age±SD: 61±7

years). Written informed consent was obtained from all subjects upon approval of the study by the

Ethic Committees of the two Institutions. All the patients were enrolled in the study immediately

after cyto-histological diagnosis when none of them had received any forms of anti-cancer therapy

whatsoever including primary surgery.

The patients underwent standard diagnostic and staging procedures consisting in a physical

examination, serum chemistry analysis, brain, chest and abdomen CT scans, radionuclide bone

scan, and bronchoscopy. The diagnosis of NSCLC was made either by bronchoscopic biopsy or

by transthoracic needle aspiration. Assessments of T and N status were based on the International

Union Against Cancer TNM staging system (25). Overall, NSCLC patients were classified as stage

I in 8 cases, stage II in 6 cases, stage III in 7 cases and stage IV in 9 cases.

At the time of enrolment into the study, twenty-seven patients were currently smokers (with

an average tobacco consumption estimated at 43 pack-years, range 20-100) while three were ex-

smokers (tobacco consumption stopped from 7.6±4.2 years) with a previous average tobacco

consumption estimated at 47 pack-years (range 20-106). Smoker patients were divided into 3

groups on the basis of tobacco consumption expressed in pack/years (group 1=<20 pack/years;

group 2=20-50 pack/years; group 3=>50 pack/years). Ex-smokers were aggregated to Group 1

considering the long time elapsing from smoking stop. Ten healthy controls were also smokers

with a mean tobacco consumption estimated at 44 pack-years.

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Patients and healthy subjects with concomitant diseases (infectious, autoimmune disease, etc) were

excluded from the study. All the patients and the healthy subjects enrolled underwent EBC and

WB collection.

EBC and WB collections

The EBC was collected by using a condenser, which allowed for the non-invasive

collection of non-gaseous components of the expiratory air (EcoScreen Jaeger, Wurzburg,

Germany). Patients and controls were asked to breathe through a mouthpiece and a two-way non-

rebreathing valve, which also served as a saliva trap, at a normal frequency and tidal volume,

wearing a nose clip, for a period of 20 min. If they felt saliva in their mouth they were instructed to

swallow it. The condensate (at least 1 ml) was collected in ice at –20 Co, transferred to 1.5ml

polypropylene tubes and immediately stored at –70 Co for the subsequent analysis.

In 10 NSCLC cases, just after collection, an aliquot of EBC was centrifuged and analysed

for cell viability by trypan-blue dye assay performed on Burker Chamber. A mean number of

83x106 cells/ml with a mean of 80% of viable cells were found. A further cytological examination

of EBC showed a lymphomononuclear origin of cells; however, most part of the cells resulted

disrupted and from the debris size they appear to be epithelial cells.

At the same time than EBC collection, a paired peripheral WB sample (3ml) was collected

from 20 healthy subjects and 28 patients; samples were put into EDTA tubes and immediately

stored at –80°C.

Microsatellite analysis

DNA was extracted from both WB and EBC by using a QIAamp DNA Mini Kit (Qiagen,

Italy), according to the “blood and body fluid protocol”. The resulting DNA was eluted in 100 µl

of sterile bidistilled water and stored at -20°C.

All the samples of the EBC DNA turned out to be positive for β-actin gene fragments.

7

EBC and WB DNA were amplified by fluorescent PCR. The analysis of microsatellite alterations

was performed using 5 polymorphic microsatellite markers from chromosome 3p which account

for hot-spots of deletions in lung cancer believed to be involved in the carcinogenesis of lung

cancer.: 3p24.2 (D3S2338), 3p23 (D3S1266), 3p14.2 (D3S1300, FHIT locus), 3p25-26

(D3S1304), 3p21 (D3S1289) (26,27). Primers’ nucleotide sequences for microsatellite analysis are

available through the Genome Database (http://www.ncbi.nlm.nih.gov/genemap-99). One of each

paired primer was fluorescent-labelled with FAM and EXE (PE Applied Biosystems ABI Prism

Linkage Mapping Set). 50 ng of DNA from EBC and whole blood were used for each PCR

amplification. PCR amplification was carried out on 10 ng of EBC and whole blood DNA in

duplicate, in a 10 µl final volume and performed on a GeneAmp 9700 thermal cycler (Applied

Biosystems) by combining the template with 0,5 U of AmpliTaq GOLD (Applied Biosystems) in

PCR buffer, 0.2 mM of each primer, 125µM of dNTPs. The PCR protocol consisted of 35 cycles

of 10 min at 94°C, 50 min at 94°C, 1 min at 52°C, 2 min at 72°C, 30 min at 60°C. Negative control

(buffer and enzyme without DNA template) was included in every PCR series. PCR products for

each clinical specimen were analysed by laser fluorescence using ABI Prism DNA sequencer (310

Applied Biosystem, Foster City, USA) equipped with GeneScan TM 2.1 software. This technique

allowed for sensitive and quantitative allele ratio estimation by measuring the peak height of both

alleles as previously described (28). Our assay is based on the detection of an alteration in the

allele ratio in the EBC DNA of healthy subjects and of patients with NSCLC when compared to

the allele ratio in the paired blood cell DNA of the same healthy subject and patient. LOH and the

presence of allele shifts indicating genomic instability were recorded in the various samples of

breath condensate and compared with the profile obtained in the DNA from blood cells. LOH was

scored when a reduction of at least 30% of allele intensity in the experimental sample was seen.

MI was defined as the appearance of clear novel band that was absent in the lane from the healthy

control blood DNA. In our systematic study, each result of amplification was confirmed by at least

2 independent analyses.

8

Statistical analysis

Fisher’s exact or chi-square tests were used to compare qualitative data. Wilcoxon Test was

used for comparison between categories. Data were expressed as means ± SD. Significance was

defined as a p value of <0.05.

9

Results

The feasibility of MA analysis in EBC-DNAs has been preliminarily evaluated. To this end,

EBC-DNA and paired WB-DNAs from 20 healthy subjects have been tested and compared for MI

and LOH analysis. Good quality DNA in terms of integrity and amount (mean quantity: 20ng/µl)

was obtained in all EBC samples. MA were found only in 7/20 EBC-DNA and in 2/20 WB-DNA

of healthy subjects. Only in EBC-DNA from one subject, a maximum of 3 MA have been shown.

Microsatellite analysis in EBC-DNA from NSCLC

We further analysed MA in EBC-DNA from 30 NSCLC patients. Also in this case, good

DNA quality in terms of integrity and amount (mean quantity: 20ng/µl) was obtained in all EBC.

Considering each microsatellite as informative when heterozygosity was evident (see M&M

section), only 19 patients gave informative results in all 5 considered loci; as regards the total

number of analysed loci (five for each of 30 patients), 90% (135/150) of the analyses resulted

informative. Four patients did not show alterations in any of the considered markers, 5 had only

one microsatellite alteration, 9 presented 4 markers contemporary altered, while none presented

simultaneous alteration in all 5 considered loci. Table 1 shows the results of the microsatellite

analysis in EBC-DNA per each locus and patient.

LOH was found in 24% (33/135) and MI in 29% (39/135) of the informative loci studied.

The most frequently altered microsatellites in EBC-DNA were D3S1300 (in 61% of the

informative DNAs for that locus) and D3S2338 (in 59% of the informative DNAs for that locus).

Microsatellite analysis in WB-DNA from NSCLC patients

When WB-DNA was considered, 28 patients had blood sample stored with 91% of the total

loci analysed resulting informative (127/140). Sixteen patients did not present alterations in any of

the studied markers, 11 had only 1 MA and none presented more than 1 locus altered.

10

Table 2 shows the results of the microsatellite analysis in the WB-DNA per each locus and

patient. LOH was found in 3% (4/127) and MI in 5.5% (7/127) the informative loci studied; the

most frequently altered microsatellites was D3S1304 (20% of the informative DNAs for that

locus).

Comparison of microsatellite analysis in EB-DNA and WB-DNA from NSCLC patients

Figure 1 shows some typical examples of MI and LOH in EBC-DNA and its paired WB-

DNA from one patient.

Data on relationships between EBC-DNA and WB-DNA were summarized in Table 3. EBC-

DNA and WB-DNA provided a similar spectrum of informative loci in all the patients (100% of

agreement in terms of capability to individualize informative loci); however, a significantly higher

number of MA was present in EBC-DNA with respect to WB-DNA (53% vs 10% of MA,

respectively; p<10-6). All MA found in WB-DNA were evident in EBC-DNA, too; conversely,

59/127 MA shown in EBC-DNA were not found in WB-DNA.

MA and clinical-pathological features

We finally analysed MA in relation to clinical-pathological characteristics of the patients.

The percentage of patients with at least one MA resulted similar in EBC-DNA from patients with

adenocarcinoma with respect to that with squamous carcinoma (86% vs 89%, respectively). In

particular, MI resulted present in a similar percentage of the 2 histotype (30% in adenocarcinomas

vs 27% in squamous carcinomas). However, LOH resulted more frequent in squamous than in

adenocarcinomas cases (36% vs 19%, respectively; p=0.05). Finally, no relationship between

number and type of MA in EBC-DNA and tumour stage was evident.

For what concerns WB-DNA, we only found a significant relationship between MA and

tumour stage; in fact, MA were present in 25% of stage I, 17% of stage II, 100% of stage III and

89% of stage IV patients (chi-square for trend p=0.02).

11

Finally, relationships with voluptuary habit were considered. Number of MA present in

EBC-DNA and increase in tobacco consumption resulted directly related either in healthy or

patient subgroups (Figure 2). In particular, the frequency of MA increased from patients of Group

I (less than 20 packs a year) to those of Group III (more than 50 packs a year) with a mean number

of MA number of 1.4+1.3 vs 3.1+0.9, respectively (p=0.02). D3S1300 locus resulted the most

frequently altered in heavy smokers NSCLC patients and this probability decreased from Group III

to Group I (from 86% in Group III to 77% in Group II to 0% in Group I).

Also differences between healthy people and patients were interesting; in fact, the number

of MA in EBC-DNA resulted significantly higher in patients of Group I than in healthy people

consuming the same amount of tobacco a year (mean number of MA: 1.4+1.3 vs 0.3+0.6,

respectively; p=0.03).

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Discussion

In the present study, the genetic alterations of microsatellites on chromosome locus 3p were

explored in EBC-DNAs from 30 NSCLC patients and 20 healthy controls. The result of this study

showed that 89% of NSCLC patients exhibited genetic alterations, either MI or LOH, in their

EBC-DNA while only 35% of the healthy subjects did so. This evidence opens interesting clinical

perspectives for the analysis of specific lung cancer genetic markers in an easily accessible and

organ-specific biological specimen such as EBC.

Some genetic events seem to trigger lung cancerogenesis and precede the morphological

transformation of cells (24). Therefore The possibility to identify genetic alterations in apparently

normal cells is the goal for an early diagnosis and optimal treatment of this tumour for which only

very weak “weapons” seem to be available today (7). Although several genetic alterations involved

in the oncogenesis of lung cancer have been candidate markers for early diagnosis, they are mainly

harvested in the cells of tumour tissue which is usually accessible only at the time of surgical

resection, i.e. when the tumour is already in an advanced phase (20,29).

The cancerization theory assumes that genetic alterations are present also in non-malignant

lung tissue adjacent to the tumour and on the entire field of the bronchial tree exposed to the

carcinogenic damages (20). For this reason molecular alterations typical of lung cancer have been

recently explored also in cells coming from the airways and collected through bronchoalveolar

lavage and induced sputum (9,23,24,29).

Recently, Gessner demonstrated the possibility of identifying genetic alterations, including

the mutations of p53 exons 5-8, in the breath condensate (8). The possibility of collecting this

sample in an easy, completely non-invasive and cheap manner, which is also well accepted by the

people, makes breath condensate a suitable tool for broader routine genetic screenings of the

population at risk and for an earlier identification of lung tumour.

13

Among the various molecular markers, a growing interest has been recently generated by

the analysis of microsatellite alterations, namely LOH and MI (23,30,31), involved in early events

and important steps of lung carcinogenesis (23).

The microsatellite alterations more related to NSCLC have been identified on the short arm

of chromosome 3 (3p) where interesting tumour suppressor genes are located, including

transforming growth factor β type II receptor (TBR II) and fragile histidine triad (FHIT) (30,31).

The allelic losses on this locus that usually result in the inactivation of these genes have been

demonstrated to be involved in tumour initiation and progression of several cancers, including that

of lung (7). The exact mechanism causing MA in lung cancer remains still unknown even if it

seems to differ from the process causing mismatch repair defects (32).

MAs have been largely studied in the DNA of the serum, induced sputum, bronchoalveolar

lavage (BAL) and tumour tissue of NSCLC patients (9,23,24,28,29,33). The frequency of the

reported MAs in lung cancer varies from 2% to 55% as a function of the specific microsatellite loci

examined and clinical pathological characteristics of the series analysed (21,22).

In this study, we have for the first time investigated the possibility to detect the

microsatellite alterations present in the EBC-DNA also comparing the obtained results with those

in paired WB-DNA. The limited presence of genetic alterations in EBC- DNAs of healthy subjects

further supports the hypothesis that this new approach could be highly specific for detection of

molecular alterations cancerogenesis-related.

Like other studies, which reported a higher frequency of LOH at locus 3p in cancer cells, a

greater percentage of MI was observed in the EBC-DNA of NSCLC patients enrolled in this study

with respect to healthy people (24,28,34,35).

As previously reported, a larger number of MAs has been observed in adenocarcinoma than

in squamous carcinoma, probably due to the association of adenocarcinoma with large-airway, in a

more direct contact with EBC (24). Conversely, in squamous carcinoma a prevalence of LOH was

found. These findings, which are in conflict with those of Park (20), who found no correlation

14

between presence of MA and histological subtype of NSCLC, seem to support the hypothesis of

Zhou, suggesting that the mechanisms of tumorigenesis could be different in various histological

lung subtypes (23).

It has long been known that the concentration of free-circulating DNA in plasma is greater

in tumour patients (28,33). This free circulating WB-DNA comes also from tumour cells although

the way through which it is released into the bloodstream remains to be definitely clarified. Several

studied have demonstrated the presence of genetic alterations in the WB-DNA of cancer patients

thus supporting the possibility of recognizing it through the use of molecular tests (33,38,39).

However, in the present study, WB-DNA has been investigated showing only 10% of MA, the

most part of them located in the same locus of the paired EBC-DNA. As expected, WB-DNA

contained fewer MA than EBC-DNA.

No significant difference in terms of percentage of MA at the different tumor stages was

observed in EBC-DNA of the breath condensate. Conversely, the percentages of MA in WB-DNA

increased as a function of tumor stage perhaps due to the amount of circulating DNA present in

blood at higher disease stages. This suggested an important role for the microsatellite analysis of

the plasma DNA not only in follow-up but also for early diagnosis of NSCLC, as suggested by

Sozzi (28,33,40). This seems to suggest a precise a role for EBC microsatellite analysis as early

marker of carcinogenesis or of susceptibility.

Recently MA has been assumed to be the expression of carcinogen exposure including

cigarette smoke (20,24,27). A high incidence of microsatellite alterations has in fact been already

reported in both former and current smokers (23,24,36). In this study, we confirm the presence of

a parallel increase of MA number and tobacco consumption already reported by the other Authors

(42,43), but, furthermore, we show that these alterations can be better detected in EBC-DNA.

These results are further corroborated by the evidence of the prevalent alteration of one loci, the

DS1300 that could have specific pathogenetic relevance. This marker is in the fragile site FRA3B

of FHIT gene (in intron 5) thus supporting the strong association between this gene inactivation

15

and carcinogenesis. Studies are ongoing to elucidate this aspect and to verify the possibility to

utilise the alterations of D3S1300 locus as maker of exposure to tobacco carcinogens. This

information seems to us one of the most important results of our study directly suggesting that our

assay performed on an airway product, i.e. EBC, could be better and direct expression of lung cell

exposure to carcinogens. Studies are ongoing on larger series of healthy people to validate the idea

that EBC could be utilised to quantify the DNA alterations due to the cumulative exposure to

smoking carcinogens.

In conclusion our results provide evidence that it is possible to investigate somatic MA in

the breath condensate DNA of NSCLC subjects. The fully non-invasiveness of breath condensate

collection and the important role of MA in lung carcinogenesis make these results potentially

relevant for the follow-up of NSCLC patients but also for the screening of high risk populations.

Further cytogenetic investigations also looking at a larger number of microsatellite markers in

patient and healthy subjects are needed to verify the potentials of these findings in terms of clinical

application.

16

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Figure legend

Figure 1: Curves obtained from fluorescent microsatellite analysis of WB and paired EBC-DNA.

Examples of reproduced MI and LOH in EBC-DNA of NSCLC patients (B=patients #1,

D=patients#23) showing microsatellite instability and different retention of heterozigosity

(D3S1300 marker) compared with unaltered matched WB-DNA (A=patients #1,C=patients#23).

The two different experiments for each patient show the reproducibility of the results.

Figure 2: Mean number of microsatellite alterations (MA) in EBC DNA of 30 NSCLC patients

and 20 Healthy divided into 3 groups on the basis of tobacco consumption. Group 1 = 0÷20

pack-years; Group 2= 20÷50 pack-years; Group 3= 50÷100 pack years. Group 1 vs group 3 in

NSCLC: p=0.02; Group1: patients vs healthy controls p=0.03. .

21

Table 1: Genetic alterations per microsatellite marker in EBC-DNAs from 30 NSCLC patients

MARKER Total

Patient IDD3S2338(3p24.2)

D3S1266(3p23)

D3S1300(3p14.2)

D3S1304(3p25-26)

D3S1289(3p21)

1 LOH LOH MI N N 3

2 H H N H H 0

3 MI H N LOH LOH 3

4 H N H H H 0

5 MI H LOH MI MI 4

6 LOH LOH H H MI 3

7 H LOH MI H LOH 3

8 H H LOH H N 1

9 N H H H N 0

10 LOH N H H H 1

11 H LOH MI LOH MI 4

12 MI H LOH MI LOH 4

13 LOH H LOH MI MI 4

14 H LOH MI H H 2

15 H H MI N H 1

16 H H LOH MI LOH 3

17 MI LOH MI MI H 4

18 H H H LOH H 1

19 MI MI MI H H 3

20 LOH LOH H MI MI 4

21 H H N H N 0

22 MI H MI H H 2

23 LOH LOH LOH H MI 4

24 H LOH H MI H 2

25 MI MI MI LOH H 4

26 MI H H MI MI 3

27 MI H MI MI LOH 4

28 LOH LOH H N H 2

29 H H N MI N 1

30 MI LOH H H H 2

H: heterozygosity; N: non informative; LOH: loss of heterozygosity; MI: microsatellite instability; Total: total number of alterations per marker

22

Table 2: Genetic alterations per microsatellite marker in WB-DNAs from 28 NSCLC patientsMARKER

Patient IDD3S2338(3p24.2)

D3S1266(3p23)

D3S1300(3p14.2)

D3S1304(3p25-26)

D3S1289(3p21)

1 H LOH H N N

2 H H N H H

3 H H N LOH H

4 H N H H H

5 H H MI H H

6 H H H H H

7 H H MI H H

8 H H H H N

10 H N H H H

11 H H H H H

12 H H H H LOH

13 H H H H H

14 H H H H H

15 H H MI N H

16 H H H MI H

17 H H H H H

18 H H H LOH H

19 H H H H H

20 H H H H H

21 H H N H N

22 MI H H H H

23 H H H H H

25 H H H H H

26 H H H MI H

27 H H H MI H

28 H H H N H

29 H H N H N

30 H H H H H

H: heterozygosity; N: non informative; LOH: loss of heterozygosity; MI: microsatellite instability

23

Table 3: Genetic alterations per microsatellite locus in paired EBC and WB-DNAs of 28 NSCLC patients. + =MA presence; - =MA absence.

EBC/Whole blood+/+ +/- -/+ -/- Total informative cases

D3S2338(3p24.2) 1 (4%) 16 (57%) - 11 (39%) 28

D3S1266(3p23) 1 (4%) 11 (42%) - 14 (54%) 26

D3S1300(3p14.2) 3 (12%) 13 (54%) - 8 (33%) 24

D3S1304(3p25-26) 5 (20%) 8 (32%) - 12 (48%) 25

D3S1289(3p21) 1 (4%) 11 (46%) - 12 (50%) 24

Total 11 59 - 57 127

Figure 1

Figure 2

0

2

Group 1 Group 2 Group 3

P=0.03

P=0.02

n=15n=9 n=14 n=3 n=7 n=2

Mea

n n.

of M

A