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Departments of Internal Medicine [J. D. M.], Pharmacology [J. D. M.], and Pathology [A. M., S. M., A. F. G.], Hamon Center for Therapeutic Oncology Research [I. I. W., J. B., C. B., A. M., N. S., J. D. M., A. F. G.], University of Texas Southwestern Medical Center, Dallas, Texas 75390; Department of Anatomic Pathology, Catholic University, Santiago, Chile [I. I. W.]; and Department of Pathology, M. D. Anderson Cancer Center, Houston, Texas 77030 [B. M.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Many mutations, especially those involving recessive oncogenes, have been described in clinically evident lung cancer (8) . Whereas some of these are common to all lung cancer types, some are more frequent in specific tumor types (8) . For risk assessment and very early lung cancer detection, it would be helpful to know the respiratory epithelial molecular events preceding the development of lung carcinoma. LOH and MA analyses using polymorphic microsatellite DNA markers are frequently used to identify allelic losses at specific chromosomal loci. Recently, we have established that allele losses at chromosomal regions on 3p usually followed by 9p21, 8p21–23, and 17p13 (TP53) losses occur relatively early during the multistage development of SQC of the lung, commencing in histologically normal and mildly abnormal (squamous metaplasia/hyperplasia) stages (5 , 9) . K-ras mutations and 3p and 9p allele losses have been detected in atypical adenomatous hyperplasia lesions in the peripheral airway (10) . However, there is no information about the genetic changes occurring in the bronchial epithelium accompanying lung ADC and SCLC.
In previous studies, we (9 , 11) and others (12) have reported a very high incidence of LOH and MAs in the normal and abnormal bronchial mucosa of cancer patients and in current and former smokers. Of interest, approximately half of histologically normal and mildly abnormal bronchial biopsies from smokers show allelic loss, and a subset of these specimens shows allele loss at multiple chromosomal sites, a phenomenon frequently observed in CIS and invasive cancer. To further understand the pathways involved in the pathogenesis of lung cancers, we compared the patterns of the molecular changes found in normal and hyperplastic epithelium accompanying SCLC with those present during the development of the other two major types of lung cancer, namely, SQC and ADC. We compared these findings with those present in the invasive component of the corresponding histological type of lung cancer.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Normal and Hyperplastic Epithelia Accompanying Lung Cancers.
We selected 30 cases representing the three major histological types of
lung cancer (10 SCLCs, 10 ADCs, and 10 SQCs) that contained multiple
noncontiguous foci of histologically normal or hyperplastic bronchial
mucosa. From these cases, we identified a total of 119 histologically
discrete foci of normal (n = 44) and hyperplastic
(n = 75) epithelia, each consisting of at least 800
cells (mean, 3.9 foci/cancer case; range, 2–5 foci/cancer case). All
epithelial specimens were obtained from centrally located large bronchi
(lobar, segmental, and subsegmental). There were no significant
differences in smoking exposure or demographic factors between the
different tumor types. Most of the bronchial epithelial samples were
taken from a different histological block than the tumor; however, the
exact spatial relationship between the epithelial samples and their
corresponding tumors could not be determined with precision.
Microdissection and DNA Extraction.
Microdissection from archival paraffin-embedded tissues was performed
either by laser capture microdissection or manually using a
micromanipulator from multiple microslides of each sample (5
, 9
, 11
, 14
, 15)
. DNA extraction was performed as described
previously (5)
. Dissected lymphocytes or stromal cells
from the same slides were used as a source of constitutional DNA from
each case. After DNA extraction, 5 µl of the proteinase K-digested
samples containing DNA from at least 200 cells were used for each
multiplex PCR reaction.
Polymorphic DNA Markers and PCR-based Microsatellite Analysis.
To evaluate LOH and MAs, we used primers flanking 19 dinucleotide
and multinucleotide microsatellite repeat polymorphisms spanning 12
chromosomal regions frequently deleted in lung cancer (8
, 9 , 16)
. The microsatellite markers and the chromosomal regions
analyzed were as follows: (a) 3p12 (D3S1274); (b)
3p14.2 at the FHIT gene (D3S4103); (c) 3p21
(D3S44623/Luca 2.2, D3S1478, and D3S1029); (d) 3p22–24
(D3S2432 and D3S1537); (e) 4p (D4S404); (f)
4q25–32 (D4S194 and D4S408); (g) 5q21–22 (L5.71 in the
APC-MCC region); (h) 6q16–21
(D6S1021); (i) 8p21–23 (D8S1130 and D8S11106);
(j) 9p21 (IFNA, D9S1748); (k) 13q14
(dinucleotide repeat at the RB gene); and (l)
17p13 (TP53 dinucleotide and pentanucleotide repeats).
Primer sequences can be obtained from the Genome Database, with five
exceptions that have been published and referenced previously
(5)
. For all samples, multiplex PCR (up to six markers)
was performed in the first amplification, followed by uniplex PCR for
individual microsatellite markers as described previously
(17)
.
Data Analysis.
Because heterozygosity at the different loci varied between subjects,
the number of chromosomal regions tested varied in subjects. Thus,
indices were calculated to compare molecular changes between
microdissected invasive lung carcinomas and epithelial samples and
between subjects (5
, 9
, 17)
. The FRL-sample index
indicates LOH for all informative chromosomal regions per
microdissected specimen (maximum, 12 regions/specimen). The FRL-subject
index indicates the LOH for all informative chromosomal regions per
subject in their bronchial epithelium specimens. The indices were
calculated as shown below.
 | (1) |
 | (2) |
To compare the incidence of MAs, we used the MA index
(17)
. The MA index indicates the MA for all microsatellite
markers per microdissected specimen (maximum, 19 microsatellite
markers/specimen) and was calculated as shown below.
 | (3) |
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. The FRL-sample
index was used to compare allelic loss at all chromosomal regions
analyzed between lung tumor types. Whereas similar high FRL-sample
index means were observed between SCLC (mean, 0.85) and SQC (mean,
0.71), ADC demonstrated a significantly lower FRL-sample index mean
(mean, 0.39; P < 0.001) in the chromosomal regions
analyzed, indicating less extensive allelic losses compared with SQC
and SCLC (Fig. 2A)
. Although
there was considerable overlap, each tumor type had a characteristic
pattern of allelic loss. SCLC and SQC demonstrated similar patterns of
allele loss for the various chromosomal regions, with three exceptions
(Table 1)
. SCLCs demonstrated higher
incidences of LOH at the 5q21–22 (69% versus 27%;
P = 0.042) and 13q14 (RB gene loci; 67%
versus 29%; P = 0.047) regions, whereas
SQCs had a higher incidence of LOH at the 8p21–23 region (92%
versus 29%; P < 0.001). In contrast, ADCs
had a distinct pattern, with lower frequencies of allelic loss than
SCLCs or SQCs at all but two chromosomal regions analyzed (Table 1)
.
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.
Most samples of bronchial epithelium accompanying SCLC (90%) had
allelic loss at one or more loci as compared with SQC (54%) or ADC
(10%). We detected a very high incidence of LOH in normal and
hyperplastic epithelium accompanying SCLCs, as expressed by a high
FRL-sample mean (mean, 0.27) and by high frequencies of LOH at any
chromosomal region (90% of the samples) compared with the other two
lung cancer types (P < 0.001; Table 1
; Fig. 2B
). Bronchial epithelial specimens obtained from SQC
demonstrated an intermediate LOH frequency (mean FRL-sample index,
0.08; LOH at any region seen in 54% of the samples) compared with
epithelial samples from ADCs (mean FRL-sample index, 0.01; LOH at any
region seen in 10% of the samples), which exhibited a significantly
(P < 0.001) lower frequency of allele loss. In fact,
several bronchial epithelial samples from SCLCs exhibited FRL-sample
indices similar to those of their corresponding tumor specimens (Fig. 2)
. The mean FRL-sample indices for tumors and epithelial specimens
were higher in SCLC than in the other cancer types. However, even after
adjusting for the relatively high tumor index, the epithelial sample
index in SCLC was relatively high, as demonstrated by the epithelial
sample:tumor ratio, which was calculated on a case-by-case basis (0.32
for SCLC, 0.11 for SQC, and 0.03 for ADC; Table 2
). No significant
differences in the incidences of allelic loss were detected between
histologically normal and hyperplastic specimens for any of the tumor
types. Higher frequencies of allelic loss were present in the bronchial
epithelium of SCLC and ADC of male patients compared with female
patients, but these gender differences were not significant.
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.
. Of interest, deletions at the 5q21–22 and 17p13
(TP53 gene) regions were never detected or occasionally
detected in epithelial samples obtained from SQCs and ADCs. However,
epithelial samples from SCLC patients had allelic loss frequencies of
32% and 63%, respectively, at these regions. In fact, the frequency
of LOH at 17p13 (63%) was the highest rate of loss for any chromosomal
region in epithelial samples from any type of cancer. SCLC-associated
epithelial samples even had higher LOH frequencies at regions
demonstrating relatively low LOH frequencies in SCLC tumors (9p21 and
8p21–23).
ASL.
Our previous studies have demonstrated that at any individual locus,
the allele loss patterns in bronchial preneoplastic lesions were not
random but had a strong tendency to be present in the same chromosomal
loci, and the identical parental allele is lost in the corresponding
invasive tumor (5
, 9
, 11)
. We refer to this phenomenon as
ASL. In cases in which nonneoplastic and tumor samples demonstrated
loss at a particular locus, we determined whether the losses involved
the same or both parental alleles. For all comparisons, allelic loss at
the same chromosomal loci was present in 54 of 94 (57%) comparisons.
We also compared the specific parental allele lost in the corresponding
nonneoplastic and neoplastic samples. For all comparisons, the same
parental allele was lost in 38 of 54 (70%) comparisons
(P = 0.001). No significant differences in the ASL
phenomenon were detected between specimens obtained from the three
major types of lung cancer analyzed.
MAs in Invasive Carcinomas and Accompanying Bronchial Epithelium.
A relatively high incidence of MAs was detected in all of the three
types of lung carcinomas (24 of 68 cases; 35%). However, there were no
significant differences in MA frequency between the histological tumor
types (Table 2)
. MAs at one or more loci
analyzed were also detected in histologically normal and hyperplastic
respiratory epithelium accompanying lung cancers (30 of 119 cases
overall; 25%). Because artifacts resulting from PCR amplification may
be mistaken for MAs, especially when minute amounts of input DNA are
used, all examples of MAs occurring in nontumor samples were confirmed
by a replicate PCR analysis. Of interest, in the bronchial epithelium,
we found a significantly higher incidence of MAs in samples
accompanying SCLCs compared with those accompanying SQCs and ADCs
(Table 2)
. No differences in the MA frequencies were detected between
normal and hyperplastic specimens. None of the MAs detected in
bronchial epithelia were identical to the alterations observed in the
corresponding invasive tumors. No gender differences in the frequency
of MAs in the bronchial epithelium accompanying lung cancers were
detected. Examples of MAs in the bronchial epithelium and invasive lung
tumors are shown in Fig. 1
.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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One of our most striking findings was the relative difference in allelic loss in bronchial epithelium accompanying lung cancers. The vast majority of samples of bronchial epithelium accompanying SCLC (90%) had allelic loss at one or more loci as compared with SQC (54%) or ADC (10%). We have previously described allelic losses in normal and mildly abnormal epithelium associated with SQC and other tumors, and higher incidences were present in more advanced preneoplastic changes [dysplasia and CIS (5 , 9 , 26) ]. The bronchial epithelium accompanying SCLC also showed a more extensive pattern of allele loss than did the other lung cancers, in some cases involving most of the chromosomal regions analyzed and demonstrating a similar or greater incidence of genetic changes than some invasive lung carcinoma cases. The finding of a very low frequency of allelic loss in proximal airway epithelium accompanying ADCs is consistent with the origin of most of these tumors from the smaller peripheral airways. The development of epithelial cancers requires multiple mutations, the stepwise accumulation of which may represent a mutator phenotype (27) . Thus, it is possible that those epithelial cells that have accumulated multiple mutations are at higher risk for progression to invasive cancer. Our findings suggest that in SCLC, in which no characteristic preneoplastic sequence of morphological changes has been described, the tumors may arise directly from either normal or hyperplastic epithelia, without passing through recognizable intermediate pathological stages (parallel theory of lung cancer development). In contrast, SQC and perhaps ADC would appear to develop after a sequence of morphological intermediate steps (sequential theory).
The multiple mutations required for cancer development that accumulate during the preneoplastic process are not random but usually follow a pattern (27) . Our present findings indicate that 5q21–22 and 17p13 (TP53 gene) deletions are frequent and early changes in the bronchial epithelium accompanying SCLC, as compared with the other lung cancer types. Recently, we have reported that allele losses at those loci are infrequent and relatively late events during the multistage development of SQC and in bronchial biopsies from smokers without cancer (5) . Mutational analysis of the TP53 gene in our SCLC cases would be desirable, but because of the very limited size of the epithelial samples, this was not feasible. We have recently demonstrated that allelic loss at three or more chromosome 4 regions is frequent in lung cancers, especially SCLC (16) . Our findings demonstrate that deletions at chromosome 4 regions may also be detected as an early event in the pathogenesis of lung carcinomas. The finding in epithelial samples from SCLC of certain genetic changes (9p21 and 8p21–23 LOH) infrequently found in the associated tumors may reflect a field effect, rather than specific changes associated with the SCLC pathway.
In previous studies, we have noted that specific chromosomal loci and specific parental alleles lost in preneoplastic lesions of the respiratory tract are usually identical to those lost in the corresponding invasive cancer (mostly SQCs), a phenomenon referred to as ASL (5 , 9 , 11 , 14 , 15) . However, in another recent study (26) , we failed to demonstrate ASL in histologically normal and hyperplastic epithelia. In our present study, which focused on histologically normal or hyperplastic epithelium, ASL was noted, but at a lower rate than reported previously in histologically and molecularly more advanced lesions.
MA (also referred to as microsatellite instability) represents changes in the size of simple nucelotide repeat polymorphic microsatellite markers, resulting in altered electrophoretic mobility of one or both alleles (17) . In lung cancers, MAs have been reported to occur at frequencies ranging from 0–45% (17) . Whereas the mechanism underlying MAs is currently unknown, they appear to represent a manifestation of genomic instability. Although SCLCs demonstrated a higher incidence of MAs than other lung cancer histological type, the differences were not statistically significant. However, the bronchial epithelia accompanying SCLC showed a significant 10-fold higher incidence of MAs compared with the corresponding epithelial specimens associated with SQC and ADC specimens. These findings are a further indication that more extensive genetic damage is present in the respiratory epithelium of patients with SCLC than in those with the other major types of lung cancer. The findings of more extensive genetic damage in the epithelial field of SCLC patients may explain the higher frequencies of second primary lung tumor in cases with this tumor type (28) .
In summary, our findings indicate that several genetic changes are common to all lung cancer histological types, whereas others appear to be tumor type specific. More extensive genetic damage accumulates during the pathogenesis of centrally located SCLC and SQC than during that of peripherally arising ADC. Our results also indicate that the most widespread and most extensive genetic damage (allelic loss and MAs) is present in the normal and mildly abnormal bronchial epithelium of patients with SCLC.
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FOOTNOTES |
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1 Supported by Specialized Program of Research
Excellence Grant P50CA70907 from the National Cancer Institute,
NIH (Bethesda, MD) and a grant from the Bristol-Myers-Squibb
Foundation Inc. (New York, NY). 
2 To whom requests for reprints should be
addressed, at Hamon Center for Therapeutic Oncology Research,
University of Texas Southwestern Medical Center, 5323 Harry
Hines Boulevard, Dallas, TX 75390. Phone: (214) 648-4921; Fax: (214)
648-4924; E-mail: gazdar{at}simmons.swmed.edu
3 The abbreviations used are: SCLC, small cell
lung carcinoma; NSCLC, non-SCLC; SQC, squamous cell carcinoma; ADC,
adenocarcinoma; CIS, carcinoma in situ; LOH, loss of
heterozygosity; MA, microsatellite alteration; FRL, fractional regional
loss; ASL, allele-specific loss.
Received 1/ 3/00; revised 3/31/00; accepted 4/ 6/00.
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