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From the Institutes of Pathology*
and
Radiobiology,
GSF-National Research Center for
Environment and Health, Neuherberg; Institute of Radiation
Biology,
Ludwig-Maximilians-University
Munich, Munich; and Institute of
Pathology§
and Department of
Surgery,¶ Technical University Munich,
Munich, Germany
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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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In 1991, Travis et al8 proposed a four-category scheme for classification of NETs including typical carcinoid tumor (TC), atypical carcinoid tumor (AC), large-cell neuroendocrine carcinoma (LCNEC), and small-cell lung carcinoma (SCLC). Although this classification system is based on light microscopic, electron microscopic, immunohistochemical, and clinical aspects of these four tumor types, investigation of genetic abnormalities in NETs may reveal additional characteristics that might be helpful in improving the reliability of prognosis and in classification of these tumors. In particular, the classification of NETs of the lung is a complex and controversial problem.10 The lack of uniform acceptance of a classification scheme has led to the proposal of several approaches.2,9,11
Several cytogenetic and molecular genetic alterations associated with
SCLC have been reported. Recently, comparative genomic hybridization
(CGH) has also been used to identify chromosomal changes in
SCLC.12-16
CGH allows the creation of copy number
karyotypes of the entire tumor genome, even from archival specimens.
Such studies, as well as conventional cytogenetic
analyses,17,18
indicate losses on chromosomes 3p, 5q, 13q,
and 17p as recurrent findings in SCLC. At the molecular genetic level,
changes have been detected in oncogenes of the myc and
ras families,19,20
as well as in tumor
suppressor genes such as p53, Rb, and the
recently identified FHIT gene.21
Additional
chromosomal imbalances included underrepresentation (losses) of
chromosomes 4q, 5q, 8q, 10q, and 15q and overrepresentation (gains) of
3q, 5p, 17q, and 19q (for reviews see Testa et al18
and
Zitzelsberger et al).22
In contrast to SCLC, to date little
is known of the cytogenetic and molecular events underlying the
development or progression of TC, AC, and LCNEC. So far, only a few
cell lines derived from TCs and ACs have been investigated by
conventional cytogenetic analysis.23-25
These studies,
however, were limited to cell lines and did not detect any
characteristic chromosomal abnormalities in these pulmonary NETs (Table 1)
. Until now, no CGH data from TC, AC,
and LCNEC are available. Several molecular studies have been performed
to elucidate the role of potential tumor suppressor genes such as
p5325-27
or Rb25,28
and
the involvement of allelic losses on chromosome 3p29
and
chromosome 1130
in the tumorigenesis of pulmonary carcinoid
tumors (Table 1)
. The importance of some of these reported findings for
tumor development is still unclear. Moreover, histogenetic relations
between phenotypically distinct NETs of the lung exhibiting different
biological characteristics are still unclear, because so far, there are
insufficient cytogenetic data available for all entities.
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The aims of this study were 1) to provide cytogenetic data on the rarely analyzed entities TC, AC, and LCNEC; 2) to identify possible genotypic relations within the entities of pulmonary NETs; and 3) to evaluate characteristic aberrations for each entity for an improved understanding of the mechanisms of tumor development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study was carried out on 29 NETs of lung from specimens
obtained from 29 patients. The histopathological classification of the
tumors was based on previously established criteria.8,10
The histopathological and clinical data from each case are summarized
in Table 2
. Follow-up information was
available on 24 cases (mean follow-up, 51 months; range, 1 to 101
months). All samples investigated were derived from formalin-fixed and
paraffin-embedded tissues. Tissue sections (10 µm) for DNA extraction
were prepared on glass slides, deparaffinized, and rehydrated.
Hematoxylin and eosin-stained sections were used to evaluate the tumor
area of the samples, which were selectively trimmed to enrich the tumor
cell content to a minimum of 80%. DNA was extracted according to
previously published protocols.31
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Metaphase spreads were prepared according to standard protocols from phytohemagglutinin-stimulated peripheral blood lymphocytes of healthy female and male donors.
CGH
CGH of labeled tumor and normal DNA was performed according to Kallioniemi et al32 and DuManoir et al33 with modifications. Isolated whole genomic DNAs (tumor DNAs and DNAs from formalin-fixed normal lung tissue) were labeled with biotin-16-dUTP using standard nick translation.34 Six hundred nanograms of biotin-16-dUTP-labeled DNA and 600 ng of SpectrumRed direct-labeled normal female or male total human genomic DNA (Vysis, Inc., Downers Grove, IL), as well as 25 µg of unlabeled Cot-1 DNA (Life Technologies, Inc., Grand Island, NY), were hybridized to denatured normal lymphocyte metaphase spreads. CGH images were captured by a black/white video charge-coupled device camera using chip integration. The three colors were digitized consecutively with specific single-color filter combinations that were automatically changed on a Zeiss Axioplan2 microscope (Zeiss, Jena, Germany). For processing of captured images, an image analysis software from MetaSystems (Altlussheim, Germany) was used. For one CGH analysis, at least 10 to 15 homologues of each chromosome were measured after 4'-6-diamidino-2-phenylindole karyotyping of 5 to 10 metaphases. Average ratio profiles were then calculated after automatically scaling the profiles of individual homologous chromosomes of the same length. Average ratio profiles were interpreted according to published criteria32,35 using statistical confidence limits based on t-statistics.
Controls
For each CGH experiment, biotinylated normal male or female DNA was hybridized against normal male or female reference DNA (SpectrumRed) as a control. Additionally, DNA was isolated from normal lung tissue of six cases and hybridized against normal reference DNA. No chromosomal changes were detected in these specimens. Three selected aberrant tumor cases were hybridized a second time by reverse labeling.
LOH Analysis
Eight polymorphic microsatellite markers along chromosome 11q were chosen to analyze 11 lung tumors and their corresponding normal tissue. These microsatellite markers consisted of D11S4936 (11q13), D11S4933 (11q13), D11S987 (11q13), D11S901 (11q14), D11S1356 (11q23.3), D11S925 (11q23.3), D11S934 (11q23 to 11q24), and D11S968 (11q25). Markers D11S4933 and D11S4936 are tightly linked to the MEN1 gene locus. Primer sequences were obtained from the genome database (http://gdbwww.gdb.org). The sense primer of each primer pair was fluorescent labeled. Microsatellite polymerase chain reaction was carried out in a total volume of 25 µl with 75 ng of isolated genomic DNA, 10 pmol of each primer, 100 µmol/L of each deoxynucleotide triphosphate, 1.5 mmol/L of MgCl2, and 1.25 U AmpliTaq Gold polymerase (Perkin-Elmer Corp., Norwalk, CT) using a Perkin-Elmer thermal cycler (system 9600). After a "hot start" (94°C for 10 minutes), polymerase chain reaction consisting of 35 cycles was performed as follows: 94°C for 1 minute; 55°C, 57°C, 60°C, and 62°C for 30 seconds each; and 72°C for 30 seconds, followed by a final extension of 72°C for 7 minutes. After visualizing the resulting DNA products on a 3% agarose gel, an appropriate dilution of each sample was loaded on a 6% polyacrylamid denaturing gel and analyzed with an automated fluorescent ABI 377 sequencing apparatus (Perkin-Elmer). Evaluation of LOH was performed as described elsewhere.36 A tumor was considered to be LOH positive, if the allele peak ratio was equal to or less than 0.6, indicating an allelic signal reduction of at least 40%. To exclude the possibility of contaminations or technical artifacts, samples were reanalyzed by independent polymerase chain reactions and gel loadings.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. They occurred with a
frequency of 4.2 aberrations per case on average (range, 0 to 11
aberrations per case). Two cases of TC (cases 18 and 21) revealed no
DNA copy number changes. The average number of aberrations increased
from 3.4 in TC (range, 0 to 9) and 4.8 in AC (range: 2 to 7) to 6.3 in
SCLC/LCNEC (range, 2 to 11). In total, 71 DNA losses and 53 DNA gains
were observed in all tumors analyzed, reflecting a predominance of DNA
losses. Moreover, no high-level amplifications were diagnosed. These
are defined by red/green ratios >2 or diagnostic profiles for
chromosomal gain detectable also by visual inspection.35
Representative profiles for typical chromosomal imbalances detected in
each entity are demonstrated (see Figure 2
).
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Chromosomal imbalances observed in 17 cases of TC are shown in
Figure 1
and Table 3
. This entity
exhibited losses on 19 chromosomes. Most frequently affected was
chromosome 11, with losses in 8 of 17 cases (47%). Four of eight cases
exhibited a deletion on 11q involving a common region on 11q13, whereas
3 of 8 cases showed the loss of one homologue of chromosome 11.
Deletions on chromosome 6q occurred in 4 of 17 cases, which represent
the second most frequent finding of DNA losses in TCs.
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ACs
Chromosomal imbalances in six cases of AC are shown in Figure 1
and Table 3
. Representative profiles for typical chromosomal imbalances
detected in AC are demonstrated in Figure 2
.
Chromosomal losses were observed on 15 chromosomes. Chromosome 11 was affected in four of six cases (66%). Again, the q arm was most frequently affected, with losses in two of six cases including the consensus region 11q13 as detected in TC. Deletions of 10q (including monosomy 10 in one case) and 13q occurred frequently in three cases of AC. The pattern of chromosomal gains was similar to the overrepresentations observed in TC.
SCLC and LCNEC
In contrast to TC and AC, a different pattern of DNA losses was
observed in 3 SCLC and 3 LCNEC cases (Table 3)
. Although 11q was
affected only in one case, 5q losses were observed in all cases.
Additional frequent DNA losses became apparent on 13q (four of six
cases), 4q (four of six cases), 3p (two of six cases), and 15q (two of
six cases). In SCLC and LCNEC, gains were detected on chromosome 19
(four of six cases), chromosome 20 (three of six cases), chromosome 5p
(two of six), and chromosome 17 (two of six cases).
LOH Analysis
We investigated eight different microsatellite loci along
chromosome 11q in 11 cases (Table 4)
. The
LOH analysis of these eight markers confirmed that this is a commonly
deleted region in TC and AC. Eight of 11 cases (73%) displayed LOH
along chromosome 11q. LOH on 11q13, affecting at least one polymorphic
marker near the MEN1 gene, was detected in 7 of 11 cases
(63%) of TC and AC. Localization of microsatellite markers, results of
LOH analysis, and comparison with CGH results for chromosome 11q are
shown in Table 4
.
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The clincopathological data for individual cases are presented in
Table 2
. Correlation with frequent cytogenetic findings (deletions on
3p, 4q, 5q, 6q, 10, 11, and 13q) were analyzed using Fisher's exact
test. The cytogenetic findings did not correlate with metastasis, death
caused by disease, sex, or smoking history (P >
0.05 for each analysis).
SCLC/LCNEC and TC/AC differ significantly for 4q (P = 0.006) and 5q (P = 0.008) deletions. A further significant difference (P = 0.002) could be observed for 11q deletions between our 23 TC/AC cases and 48 SCLC cases from the literature.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In contrast to SCLC, TC as well as AC and LCNEC
have only rarely been investigated for chromosomal aberrations. To our
knowledge, this is the first CGH study on these entities of pulmonary
NET. Our CGH findings strongly suggest that distinctive chromosomal imbalances occur in the various subgroups, allowing a cytogenetic discrimination between TC/AC and SCLC/LCNEC. The most striking difference are underrepresentations on chromosome 11q, which are frequent in TC (8 of 17) and AC (4 of 6) but rare in SCLC (0 of 3) and LCNEC (1 of 3). For statistical analysis we used published CGH data on 45 cases of SCLC13-15 in addition to our own CGH results on three cases of SCLC. Statistical analysis reveals a significant difference (P = 0.002; Fisher's exact test) in the occurrence of 11q losses involving chromosomal region 11q13 between TC/AC (10 of 23; our cases) and SCLC (4/48; published cases and our cases). Thus, TCs and ACs are both characterized by underrepresentation of 11q, but ACs show further losses on 10q and 13q.
A first indication of 11q aberrations in pulmonary carcinoid tumors came from recent LOH studies.30,37 Based on these LOH studies, it was hypothesized that chromosomal losses in the MEN1 gene-containing region might be significant in the pathogenesis of TC and AC, both associated and not associated with the multiple endocrine neoplasia type 1 (MEN1) syndrome. The MEN1 locus was previously localized to chromosome 11q1338-41 and was recently cloned.42 A recent study of sporadic lung carcinoid tumors showed two inactivated copies of the MEN1 gene in 4 of 11 cases.43 These findings are now supported by our CGH data, which implicate DNA losses on chromosome 11q with the pathogenesis of sporadic lung NETs, representing a characteristic cytogenetic alteration in these tumors.
To date, 10q deletions have been reported for endometrial carcinomas,44 malignant meningiomas,45 gliomas,46 and prostate carcinomas47 and have been associated with tumor progression. For the much more aggressive SCLC, 10q losses have also recently been reported.13-16 These finding in SCLC provide a clue for the MXI1 gene located on 10q24–25 as a potentially affected tumor suppressor gene, given that its function as a negative regulator of myc oncogenes coincides with frequent amplification and overexpression of myc oncogenes in advanced SCLC.19,20 MXI1 has also been suggested to act as a tumor suppressor in prostate cancer.47 13q losses in AC include the RB1 locus, which is a well known tumor suppressor gene, loss of which is associated with tumor progression and poor prognosis in several tumor types. LOH in the RB1 locus has previously been detected in 92% of SCLCs, 80% of other neuroendocrine carcinomas, and 33% of carcinoid tumors.28
Our three cases of LCNEC, representing a rare pulmonary tumor, exhibit similar chromosomal changes to our three SCLC cases. The chromosomal imbalances include preferential losses of chromosomes 4q, 5q, 13q, and 15q, which is in accordance with previously published CGH data from SCLC. We did not observe loss of chromosome 17p in any of the SCLC and LCNEC cases reported in this study. This possibly reflects the rather early tumor stages of our cases (pT1 and pT2). 17p losses were also absent in our subset of TC and AC, which is in good accordance with recent p53 studies of typical and atypical carcinoids, which did not show any p53 mutations.27 However, Lohmann et al26 reported mutations of the p53 gene in pulmonary carcinoid tumors at a low frequency (4/25), whereas they occurred in more than 80% of the 27 SCLC cases they investigated.48
Loss of the chromosomal region 5q13–21 has frequently been described in SCLC,12-16,49 and candidate tumor suppressor genes APC and MCC have been mapped to this region. 3p deletions are also very common in SCLC and have been demonstrated in several cytogenetic and molecular genetic studies identifying distinctive deleted subregions on 3p.17,18,12,13,15,16,50,51 They co-localize in our SCLC cases with chromosomal regions potentially harboring tumor suppressor genes such as the FHIT gene on 3p14.2,52 which was reported to exhibit deletions on several exons in 80% of SCLCs.21 Our CGH results on TC and AC suggest that 3p deletions are rare events in these tumors (1 of 17 TCs and 1 of 6 ACs). Previous cytogenetic studies of a few pulmonary carcinoid tumors did not detect any 3p deletions,23,24 except for that of Lai et al,25 who reported chromosomal abnormalities of 3p in four of four pulmonary NETs.
We confirmed our CGH results by LOH studies of microsatellite loci
along 11q in 11 TC/AC (7 cases with 11q deletion and 4 cases without
11q deletion for controls). LOH findings on 11q loci are in good
accordance with our CGH results (Table 4)
, except for two cases (cases
18 and 27, Table 4
) with LOH in D11S4933 and D11S901, which exhibit
either DNA loss in another chromosomal region (case 27) or no DNA loss
on 11q (case 18). This can be explained by the extent of the deleted
segment, which is possibly below the detection limit of CGH.
In this investigation we found no relationship between chromosomal DNA changes and clinical parameters as survival, metastasis, sex, or smoking history. This may be attributed to the number of cases in each tumor group, which is likely too small to show clear statistical correlations between clinical and CGH findings.
Our CGH results from TC, AC, and LCNEC together with published CGH data on SCLC characterize chromosomal aberration patterns for each subgroup with a predominant occurrence of 11q losses in TC and AC and different aberration patterns in SCLC and LCNEC. Therefore, it appears that carcinoid tumors and the high-grade tumors have substantially different chromosomal changes that could be explained either by different progenitor cells or differences in carcinogen exposure, such as cigarette smoking. Strong epidemiological differences between lung carcinoid tumors and SCLCs indicate a markedly different process of carcinogenesis, which casts doubt on the hypothesis of a common cell precursor.53,54
Although NETs of the lung share common histomorphological features, they differ greatly in their cytogenetic characteristics, highlighting a fundamental molecular divergence between these tumors.
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Acknowledgements |
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Footnotes |
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Accepted for publication July 18, 1998.
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References |
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