111

Towards a genetic-based classification

of human lung cancer

Iver Petersen ∗ and Simone Petersen 1. Introduction: Concepts in current classification

Institute of Pathology, University Hospital Charité,
Lung cancer represents the most important chemi-
Berlin, Germany cally induced tumour type in man since the vast ma-
jority of lung cancer are associated with long term
Received 4 October 2000
cigarette smoking which is true for all major subtypes.
Accepted 11 December 2000
The latency is several decades and most carcinomas
develop after the age of 50 with the highest incidence
Lung cancer is a highly aggressive neoplasm which is re- around the age of 60. Although there is probably a sub-
flected by a multitude of genetic aberrations being detectable group of smokers who better tolerate the carcinogen
on the chromosomal and molecular level. In order to under- the risk is steadily rising with age and still many pa-
stand this seemingly genetic chaos, we performed Compar-
tients become symptomatic within the 70-ties or 80-
ative Genomic Hybridisation (CGH) in a large collective of
ties [9].
human lung carcinomas investigating different tumor enti-
ties as well as multiple individual tumour specimens of sin- The major classification schemes of lung cancer are
gle patients. Despite the considerable genetic instability be- represented in Table 1. The new WHO classification
ing reflected by the well known morphological heterogeneity defines 9 subgroups of malignant epithelial lung tu-
of lung cancer the comparison of different tumour groups us- mours of which the major subcategories are squamous
ing custom made computer software revealed recurrent aber- cell carcinoma, adenocarcinoma, large cell carcinoma
ration patterns and highlighted chromosomal imbalances that and small cell carcinoma [32]. The authors also present
were significantly associated with morphological histotypes the spectrum of neuroendocrine lung tumours ranging
and biological phenotypes. Specifically we identified imbal- from the typical carcinoid, the atypical carcinoid to the
ances in NSCLC being associated with metastasis formation highly aggressive neuroendocrine carcinomas consist-
which are typically present in SCLC thus explaining why ing mainly of LCNEC and SCLC. This concept sug-
the latter is such an aggressive neoplasm characterized by
gests that there is a transition from the more benign
widespread tumor dissemination. Based on the genetic data a
carcinoid tumours to the highly malignant SCLC. In
new model for the development of SCLC is presented. It sug-
gests that SCLC evolving from the same stem cell as NSCLC other classifications this has lead to the designation
should be differentiated into primary and secondary tumors. of neuroendocrine tumors (NET) for the benign tu-
Primary SCLC corresponding to the classical type evolved mours and neuroendocrine carcinoma (NEC) for the
directly from an epithelial precursor cell. In contrast, sec- malignant ones. It still seems to be based on the old
ondary SCLC correlating with the combined SCLC develops assumption that SCLC and LCNEC are derived from
via an NSCLC intermediate. In addition, we established li- a neuroendocrine precursor cell, i.e., the Kulchitsky
braries of differentially expressed genes from different hu- cell, whereas NSCLC develop from an epithelial pre-
man lung cancer types to identify new candidate genes for cursor cell. However, these transitions are very rarely
several of the chromosomal subregions identified by CGH. observed. Instead there is evidence that SCLC and
In this review, we summarise the status of our results aiming NSCLC may be derived from the same cell type, some-
at a refined classification of lung cancer based on the pattern
times called the amphicrine stem cell since it may give
of genetic aberrations.
rise to a neuroendocrine as well as epithelial differen-
tiation [10].
* Corresponding author: Iver Petersen, MD, Institute of Pathology, Whereas the major subtypes adenocarcinoma, SCC,
Charité, Humboldt-University, Schumannstrasse 20-21, D-10098 LCC and SCLC are relatively easy recognisable it is
Berlin, Germany. Tel.: +49 30 2802 2611; Fax: +49 30 2802 3407; fairly difficult to differentiate some variants from each
E-mail: iver.petersen@charite.de. other. For instance, many pathologist will have diffi-

Analytical Cellular Pathology 22 (2001) 111–121

ISSN 0921-8912 / $8.00  2001, IOS Press. All rights reserved
112 I. Petersen and S. Petersen / Lung cancer classification

Table 1
initial phase. In contrast, it is far more difficult to pre-
Classification schemes of malignant epithelial lung tumors
dict the outcome of NSCLC based on morphological
Actual WHO classification grounds. There are tumours similar to SCLC with a
1. Squamous cell carcinoma (SCC) highly aggressive behaviour and early metastasis for-
Variants: papillary, clear cell, small cell, basaloid SCC mation, whereas others may be cured after surgery or
2. Small cell carcinoma (SCLC) remain stable for a considerable period of time even in
Variant: combined SCLC the case of residual disease.
3. Adenocarcinoma (Adeno) In this review we describe our strategy and results
Variants: acinar, papillary, bronchioloalveolar, solid, in the attempt to contribute to the understanding of hu-
mixed, others man lung cancer genetics and why we believe that this
4. Large cell carcinoma (LCC) knowledge will have a major impact on a refined clas-
Variants: LCNEC, basaloid, lymphoepithelioma-like, sification of the disease.
clear cell, with rhabdoid phenotype
5. Adenosquamous carcinoma
6. Carcinoma with pleomorphic, sarcomatoid or sarcoma-
tous elements
2. CGH results
7. Carcinoid tumors
Variants: typical, atypical
Comparative Genomic Hybridization (CGH) is a
8. Carcinomas of salivary-gland type
molecular cytogenetic technique for the detection of
9. Unclassified carcinomas
DNA gains and losses [5]. As a screening method it
Spectrum of neuroendocrine lung tumors
provides a survey of the entire tumor genome. DNA
Typical carcinoid – Atypical carcinoid – LCNEC – SCLC
overrepresentations are potentially associated with the
Major clinical differentiation
activation of proto-oncogenes while deletions might
NSCLC (Adeno, SCC, LCC) ↔ SCLC
indicate the inactivation of a tumor suppressor gene.
For conventional CGH, however, the resolution is lim-
Abbreviations: LCNEC, large cell neuroendocrine carcinoma,
NSCLC, non-small cell lung carcinoma (other abbreviations as men- ited to approximately one chromosomal band allow-
tioned in the table). ing the detection of deletions in the order of 10 Mb
whereas for amplifications a size of 2 Mb (product
culties to subdivide large cell carcinomas into the four of amplicon size and copy number) is achievable [1].
possible subgroups, i.e., classical LCC lacking neu- Thus any correlation with the status of a gene within
roendocrine morphology and neuroendocrine differen- a specific chromosomal region needs to be confirmed
tiation accessed by immunohistochemistry and/or elec- by additional studies. Recently new approaches have
tron microscopy, LCC with neuroendocrine differenti- been published which will extend the resolution of
ation lacking neuroendocrine morphology, LCC only CGH [24,30]. However, the majority of CGH data now
with neuroendocrine morphology and finally LCNEC arising in the literature is still gathered by the classical
showing both characteristics. techniques using normal chromosome spreads as the
Although not explicitly stated in the WHO classi- DNA matrix to which the genomes bind.
fication SCLC actually consists of two variants, one In the recent years we analyzed a collective of lung
might be termed the classical type showing either the carcinomas by CGH comprising more than 250 tumour
typical oat cell or fusiform morphology and the com- specimens. The data has either been published [13,
bined SCLC presenting as a mixture of an NSCLC 14,18,29,33] or is available at our CGH online tu-
and SCLC. In the clinical setting the major distinc- mor database at http://amba.charite.de/cgh. Beside pri-
tion is between SCLC (∼20% of all lung cancers) and mary tumors also metastases and tumor cell lines
NSCLC (∼80%). The latter may show either the pure were analyzed. The tumour DNA were mainly ob-
differentiation of one of the major types (SCC, Adeno, tained from frozen tissue derived from surgical re-
LCC) or a mixed differentiation which is a frequent sections at the Department of Surgery of the Char-
finding due to the high percentage of morphologically ité Hospital at the Humboldt-University Berlin. Addi-
heterogeneous tumours [11]. The histopathological di- tionally, snap frozen tumour specimens of primary and
agnosis of SCLC is highly predictable for the clinical metastatic lesions were collected at post mortem ex-
course which is characterised by a highly aggressive aminations.
phenotype with early and widespread tumor dissemi- Based on our own CGH software [27] we extended
nation and excellent response to chemotherapy in the the functionality of conventional CGH programs by
 I. Petersen and S. Petersen / Lung cancer classification 113

taking advantage of the fact that the primary data in tions or high copy amplifications, respectively. These
CGH being derived from digital analysis of fluores- alterations are frequently observed in SCLC and corre-
cence images is already computerised to calculate his- spond to the observation that the tumour often harbours
tograms and difference histograms [14]. There are two amplifications [3].
major advantages of this approach. First the number Our relatively small study on neuroendocrine lung
of tumour samples that can be simultaneously visu- tumors showed that LCNEC has similar alterations as
alised and analysed is virtually unlimited. Second and SCLC, particularly the deletions of 3p and 10q were
even more important is the fact that allows the statisti- frequently found. Interestingly, the CGH patterns of
cal comparison of tumour subgroups highlighting dis- carcinoids and neuroendocrine carcinomas were not
tinct chromosomal subregions of which the difference similar [33].
in gains or losses between these subgroups is statisti- NSCLC showed overlapping as well as different al-
cally significant. terations compared to SCLC (Fig. 1B). They also car-
The importance of the DNA gains and losses de- ried a high incidence of deletions on chromosomes 3p,
tected by CGH are underlined by the fact that kary- 4, 5 and 13q. In addition DNA gains occurred at a
otyping and more recently M-FISH and SKY analy- high frequency on chromosomes 1p, 6q, 9p, 18q and
sis failed to detect recurrent translocations in lung can- 21q. Also for the common chromosomes the patterns
cer [31]. Thus in contrast to leukaemia, lymphoma and are slightly different. For instance, chromosome 3p is
sarcoma, oncogenic fusion protein do not seem to play particularly affected by interstitial deletions whereas in
an important role in solid tumours. SCLC the entire chromosome arm or large regions of
it are affected. For chromosome 13q, SCLC showed
2.1. Comparison of SCLC and NSCLC deletions of the proximal arm including the locus of
the Rb gene at 13q14 whereas in NSCLC typically the
Figure 1A shows the histogram of SCLC. The SCLC distal chromosome arm is lost.
collective comprised the previously published autopsy The above mentioned differences are best visu-
cases [13,26,29] and several additional samples includ- alised by the difference histogram between SCLC and
ing 7 tumour cell lines. In general, the results of the NSCLC shown in Fig. 1C. It clearly indicates that
autopsy cases were similar to those of the cell lines the deletions of the entire chromosome 3p, 10, 4p16,
suggesting that SCLC cell lines constitute a valuable 15q, 16q, 17p as well as the overrepresentations on
model to study this tumour type. The typical findings chromosomes 1, 3q, 6, 13, and 17q24–q25 are signif-
in SCLC are deletions on chromosomes 3p, 4, 5q, 10q, icantly associated with SCLC. In contrast, the dele-
13q, 17p and DNA gains on 3q, 5p, 6p, 8q and 17q. tions of chromosome 1p, 6q, 9p, 18q, 21q and the gain
Interestingly, deletions are more frequent than DNA of chromosome 22q is significantly more frequent in
gains suggesting that the inactivation of tumour sup- NSCLC.
pressor genes is as important as gain of function muta-
tions of proto-oncogenes. 2.2. Comparison of non-metastatic and metastatic
SCLC usually harbour large deletions affecting en- NSCLC
tire chromosome arms or whole chromosomes as ex-
emplified by the deletions on 3p, 10 and 17p. Deletions In a recent study we examined primary SCC with-
of 3p have been reported as the characteristic finding out evidence of tumor dissemination, i.e., stage pN0
in SCLC [35]. Since other tumour types also carry a and pM0, with metastasising carcinomas showing
high incidence of DNA losses on 3p the deletion per hematogenous (pM1) and/or lymphatic tumour spread
se can not be considered specific. However, there is (pN+). For most chromosomal regions the latter tu-
a typical pattern regarding chromosome 3 in SCLC mour group harboured more alterations which is con-
which consists of the deletion of the entire short chro- sistent the paradigm of tumour genetics postulating
mosome along with the overrepresentation of the long that tumour progression and metastasis formation is
arm, i.e., the CGH equivalent of a 3q isochromosome. characterised by an accumulation of genetic defects.
The 3p deletion and the 3q gain often fulfil the crite- Specifically, the deletions at 3p12–p14, 4p15–p16, and
ria of pronounced imbalances being defined as those 10q as well as the gain on chromosome 1q22–q25 were
imbalances with a ratio exceeding the thresholds of associated with the metastatic phenotype [18]. In an-
0.5 and 1.5 [18]. The pronounced losses (ratio < 0.5) other study of 42 brain metastases we additionally ob-
and gains (ratio > 1.5) are related to multi copy dele- served a peak in the histogram for the gain at 17q24–
114 I. Petersen and S. Petersen / Lung cancer classification

Fig. 1. Histogram of SCLC (A) and NSCLC (B). The chromosomal imbalances are shown as incidence curves along each chromosome. Areas
on the left side of the chromosome ideogram correspond to loss of genetic material, those on the right side to DNA gains. The frequency of
the alterations can be determined from the 50% and 100% incidence lines depicted parallel to the chromosome ideograms. DNA changes with
99% significance are colored in black, additional changes with 95% significance are depicted in light gray. The proportion of pronounced DNA
imbalances are visualised in dark gray (areas close to the 0% incidence line). They are most likely to represent high copy amplifications or
multi copy deletions. (C) Difference histogram of SCLC and NSCLC. Green, percentage of changes that are exclusively present in NSCLC.
Dark gray, excess of changes in SCLC. White areas beneath the colored parts of each histogram, percentage of changes that are present in both
tumour-subgroups. Grey horizontal lines, statistically significant differences. Light grey lines, regions with 95% significance; dark grey lines,
99% significance according to the χ2 -test.
 I. Petersen and S. Petersen / Lung cancer classification 115

Fig. 1. (Continued).

q25 suggesting that the amplification of a gene at this primary tumour. This offers an important outlook for
chromosomal regions might mediate tumour dissemi- a refined new classification since small biopsies might
nation into the nervous system [19]. Interestingly, these be used to determine the malignant potential. Similar
alterations were also associated with the SCLC pheno- to morphological grading such a genetic grading must
type providing a genetic correlate to the fact that SCLC be correlated in its predictive value to conventional tu-
is a highly metastatic tumour type which often spreads mour staging which still constitutes the gold standard
into the brain. to describe the status quo of the biological tumour phe-
The statistical analysis of the tumour subgroups notype. However, our recent analysis of primary head
(pM0/pN0 SCC versus pM1/pN+ SCC) was supple- and neck squamous cell carcinomas, the first tumor
mented by the analysis of primary and corresponding collective where patients survival data was available,
metastatic tumours. Although the tumours of the same indicated that the analysis of the chromosomal imbal-
patients harboured a high percentage of common im- ances are even a better prognostic indicator than pTNM
balances indicating the clonal relationship the compar- staging [2]. This is a very encouraging result.
ison also revealed a considerable heterogeneity indicat- Finally the comparison of the primary SCC with
ing the genetic instability of lung cancer on the chro- their corresponding metastases also suggested certain
mosomal level. Individual imbalances that were indi- mechanisms of the clonal evolution of chromosomal
cated by the statistical analysis of the tumour groups changes during tumour progression. Overrepresenta-
could be additionally found in metastatic lesions. How- tions were reduced in size. In particular, this was ob-
ever, many of them were already detectable the pri- served for gain of chromosome 1q which resulted in
mary tumour similarly to our findings in primary and the overrepresentation of the centromeric region 1q21–
metastatic SCLC [29]. There are two main conclusions q25 constituting exactly the region putatively harbour-
from this. First, the comparison of tumour subgroups ing a proto-oncogene of relevance in metastasis forma-
seems more appropriate to dissect the genetic alter- tion. In contrast, small interstitial deletions were of-
ations that are responsible for a tumour phenotype than ten extended to the loss of entire chromosome arms or
the comparison of individual tumours since the latter even whole chromosomes which was seen for several
type of analysis is largely biased by the genetic tumour regions, e.g., chromosome 10q [18].
heterogeneity. Second and even more importantly, the The analysis of different types of NSCLC indicated
potential of a tumour for hematogeneous spread can at chromosomal imbalances that were associated with tu-
least partially be deduced by the genetic analysis of a mour differentiation. Adenocarcinomas, for instance,
116 I. Petersen and S. Petersen / Lung cancer classification

are typically characterised by overrepresentations of 3. Model of primary and secondary SCLC

chromosome 1q. This again provides a genetic cor-
relate to the fact that adenocarcinoma carry a higher The above mentioned CGH results indicate that
risk for hematogenous metastasis formation than lung SCLC and NSCLC are genetically related tumour en-
SCC [14]. tities and that NSCLC have the potential to evolve into
a SCLC during tumour progression. Obviously the two
components of combined SCLC are not genetically in-
2.3. Morphological tumour heterogeneity: analysis of dependent. Rather the morphological heterogeneity is
a combined SCLC a detectable correlation to the genetic instability of this
tumour type and lung cancer in general. A model for
the development of SCLC which is based on the over-
A metastasizing combined SCLC was analysed after lapping CGH patterns of metastatic SCC and SCLC,
microdissection. The primary tumour showed a squa- the chromosomal mechanisms during tumour progres-
mous cell differentiation together with a SCLC compo- sion as well as the analysis of the combined SCLC
nent (Fig. 2A). A synchronous lung metastasis showed is depicted in Fig. 3. The primary SCLC correlating
exclusively the SCLC phenotype (Fig. 2B). Not sur- with the classical SCLC develops directly from a pre-
prisingly, CGH revealed a clonal relationship between cursor cell of probably epithelial origin. In contrast,
both tumours as shown in Fig. 2C and 2D, respectively. the secondary SCLC evolve via a NSCLC intermedi-
Thus, the SCLC must have evolved from the SCC com- ate. The secondary SCLC thus correlates with the com-
ponent of the tumour. It is interesting to note, that bined SCLC. It is important to note that it is also a
the expression of the neuroendocrine marker Synapto- model of lung cancer in general in which small cell
physin and Chromogranin was restricted to the SCLC and neuroendocrine differentiation should be consid-
component while the SCC was negative. ered as markers for tumor progression. In contrast we

Fig. 2. H&E stains of the primary combined SCLC showing of a SCC component (A) and a synchronous metastasis exclusively with the SCLC
phenotype (B). After microdissection the SCC component of the primary tumour (C) and the SCLC metastasis (D) was analysed by CGH showing
a clonal relationship as evidenced by the high number of common changes.
 I. Petersen and S. Petersen / Lung cancer classification 117

correlation with the CGH data. Using allelotyping,

we confirmed that deletions of this chromosome arm
are a typical finding in SCLC and that it is associ-
ated with tumour progression and metastasis formation
of lung SCC. We identified three minimal regions of
deletions putatively harbouring the tumour suppressor
genes. However, any of three major candidate genes,
i.e., MXI1, PTEN/MMAC1 and DMBT1, were not af-
fected in lung cancer [15,16,20].
Our CGH results is also in excellent agreement with
the data on the prevalence of specific gene defects in
different lung cancer types published by others and us.
For instance, SCLC typically show a high incidence
Fig. 3. Model of primary and secondary SCLC.
of deletions at 17p13 and 13q14 correlating with the
strongly feel that the presence of neuroendocrine pro- observations that TP53 and RB1 are frequently inac-
teins should not be used as an indicator of a putative tu- tivated in this tumour type. In NSCLC, the high inci-
mour stem. Of course, these consideration apply only dence of DNA gains at 11q13 reflects the fact that cy-
for lung carcinomas and not for carcinoids or benign clin D1 gene is frequently amplified [6,25,28,36].
neuroendocrine tumours of the lung. In summary, the CGH data clearly indicate the fea-
The model strengthen the notion that NSCLC and sibility of a genetic lung tumour classification with the
SCLC are derived from the same stem cell and that potential to provide superior results to morphological
both tumour types are more closely related to each characterisation, in particular for the possible assess-
other than SCLC with other neuroendocrine lung ment of the metastatic potential.
tumors, in particular carcinoids. Epidemiology and There are however some disadvantages that will
pathology also support this view. Cigarette smoking is probably prevent CGH from becoming a routine met-
the typical risk factor for SCLC and similar to NSCLC, hod in genetic tumour classification. As mentioned
dysplasia and preneoplastic epithelial changes are a above, the method has a limited resolution. In addition,
common finding. In contrast, neuroendocrine precur- there is a considerable work load associated with the
sor lesions are only very rarely observed. Thus, for analysis of a single tumour [12]. First the preparative
the majority of cases the neuroendocrine carcinomas steps, i.e., DNA extraction, labeling and hybridization,
SCLC and LCNEC should be regarded as distinct tu- take several days. Second, image capture and anal-
mor types than carcinoids being derived from different ysis and in particular the careful karyotyping of the
stem cell. Although there may exist the progression of 15 metaphases that we usually analyse takes several
a typical to an atypical carcinoid and finally a SCLC, hours. Thus, in an optimistic estimate the final result
the progression of a NSCLC to a SCLC is probably will be available after one week with one sample per
much more frequent. Therefore, SCLC might be re- day and technician. In comparison, conventional pro-
garded as an end stage tumour with the most aggressive cessing of the tumour specimen and histopathological
phenotype of all lung carcinomas. The situation is sim- evaluation is much faster and cheaper. Therefore it is
ilar to glioblastoma multiforme of the brain. Mostly it an important question whether a laborious CGH anal-
primarily presents as a glioblastoma but in some cases ysis can be replaced by other methods.
first an astrocytoma is diagnosed which later progress Copy number changes and loss or gain of func-
to the most malignant brain tumor in man. Interest- tion mutations are frequently associated with either re-
ingly, glioblastomas and SCLC also have some chro- duced expression or overexpression of tumor associ-
mosomal alterations in common, in particular the loss ated genes. We therefore performed an immunohisto-
of chromosome 10 [34].
chemical analysis of the HER/NEU proto-oncogene to
answer the question whether the DNA gain on chromo-
4. Correlation between chromosomal changes and some 17q21 where the gene is located correlates with
genetic defects protein overexpression [8]. As depicted in Fig. 4, this
was indeed the case. Similar to breast carcinomas the
For chromosome 10q, we performed extensive ad- laborious genetic analysis can thus be largely replaced
ditional genetic analysis which showed an excellent by a simple immunohistochemical test.
118 I. Petersen and S. Petersen / Lung cancer classification

Table 2
Results of the SSH analysis
Libraries (SCLC, SCC, Adeno) 6
Total number of clones 2471
Already sequenced 1554
Known genes 49% (40–60%)
Expressend sequence tags 39% (24–60%)
Unknown sequences 12% (10–16%)
Analyzed by Northern blot 363
Differentially expressed 73.7% (54–92%)

driver and then as the tester two libraries are generated

that will represent mainly the genes that are overex-
pressed and underexpressed within the tumour, respec-
tively. We meanwhile generated six of such libraries
by comparing an adenocarcinoma, a SCLC and a SCC
with normal bronchial epithelial cells [21]. A compila-
tion of the data is given in Table 2.
Fig. 4. The overexpression of HER2/NEU being located at chromo- It is important to note that more than 10% of the
some 17q21 correlates with DNA gains of the respective chromoso- clones are not represented in the public databases.
mal band in NSCLC. About 40% correspond to so called expressed sequence
tags which are cDNA fragments of genes of which the
Thus, the chromosomal imbalances are most prob- full length cDNA has not yet been determined and the
ably associated with a distinct pattern of gene expres- function is unknown. Although approximately half of
sion mediating the biological phenotype of the tumor. the clones represent known genes being listed in the
This hypothesis is strongly supported by recent cDNA Unigene set of the NCBI database their importance in
microarray analysis of breast carcinomas [23]. tumorigenesis of lung cancer is mostly unknown.
Based on our negative experience with the candidate We are performing Northern blot analysis to con-
genes on chromosome 10q and taking into considera- firm the expression pattern of the cDNA clones [21].
tion that many tumour associated genes are either not Although this technique is laborious and might appear
yet identified or fully characterised we sought for alter- somehow old-fashioned in view of the modern screen-
native methods to rapidly identify new candidate genes ing methods like cDNA arrays it has several impor-
in lung cancer. tant advantages. First, Northern blotting is still the gold
standard for the assessment of gene expression on the
RNA level. For our libraries, it indicated that more
5. Identification and characterisation of new than 70% of the clones are indeed differentially ex-
candidate genes pressed. Second, it provides a measurement for the size
of the full length RNA transcripts which is essential to
5.1. Generation of cDNA libraries of differentially know if the entire cDNA needs to be isolated. Third,
expressed genes by including additional RNA samples in the Northern
blot analysis the frequency of either reduced or en-
Abnormal gene expression is a hallmark of the neo- hanced gene overexpression in the tumor type can be
plastic phenotye. Therefore we used expression genet- estimated. We meanwhile analyze at least RNA sam-
ics to identify new candidate genes in lung carcino- ples from 3 tumor cell lines from a SCLC, SCC and
genesis. Specifically we applied the method Subtrac- adenocarcinoma by this procedure. Although it is too
tive Suppression Hybridziation (SSH) to generate li- early to draw a final conclusion on the patterns of gene
braries of differentially expressed genes [4]. Without expression in SCLC and NSCLC the fact that several
going into detail the technique is based on the subtrac- genes are represented in the different libraries and that
tion hybridisation of so called tester cDNA versus a many show a similar expression pattern in the North-
driver cDNA which results in the enrichment of clones ern blot analysis supports the above mentioned model
of either cDNA pools. By using the tumour first as the of primary and secondary SCLC.
 I. Petersen and S. Petersen / Lung cancer classification 119

Table 3
Overexpressed genes identified by SSH
Protein type Protein name
Oncogenes N-myc, c-myb, Ki-ras2, N-ras-related gene unr
Receptor tyrosine kinases Epidermal growth factor receptor EGFR,
homolog to the ERBB2 receptor tyrosine kinase
SH2 -domain proteins Grb7, Grb14
Cell–cell adhesion molecules Intercellular adhesion molecule I ICAM-I
Cytosolic proteins Stathmin Op18, Ran-binding proteins 1 and 5, centrosome-associated kinase BTAK
Growth factors/cytokines Glia maturation factor β GMFB, interleukin 8
Cytoskeletal proteins Zytokeratin K18, actin-bundling protein L-plastin
DNA interaction Topoisomerase 1 and 2α
Regulatory proteins E1A binding protein p300, translation initiation factor INT6, replication factor C
Surface molecules, antigens Epithelial membrane protein 1 EMP1, carcinoembryonic antigen 1 CEA1,
NY-CO-1, NY-CO-25, epithelial glycoprotein EGP
Ca++ -dependent enzymes Calpain, calcyclin-binding protein
Energy metabolism Cytochrome c, cytochrome b, mitochondrial proteins

Table 4
Underexpressed genes identified by SSH
Protein type Protein name
Cell adhesion and communication
Extracellular matrix proteins Laminin α 3a, fibronectin, thrombospondin 1
Cell membrane associated proteins CD44-variant, integrin α6, integrin β1, connexin 26, tissue factor, desmocollin Typ4, fibronectinrecep-
tor, bullous pemphigoid antigen 1, desmoplakin1 and 2, oncostatin M-receptor
Protease inhibitors Cystatin A, maspin, plasminogen-activator-inhibitor 2, VATKI, SSCA1 epidermal differentiation com-
plex, small proline-rich proteins 1 and 3, cornifin B
Ca++ -binding proteins CaN19, MRP-8/calgranulin A, calgranulin B, Ca++ -activated chloride channel protein 2 and 3
(CaCC2,3)
Cytoskeleton protein
Cytokeratins K6A, K6, K15
Others β-γ-non-lens-crystallin AIM1, moesin
ERM-binding phosphoprotein 50
Regulatory proteins
Transcription factors p51B/p73H, CUSP (member of the p53-family)

The known genes belong to very different classes of be soon accessible. Thus our procedure is exemplary
proteins, e.g., being involved in cellular signaling, the for future gene discovery.
cytoskeleton and the interaction with the extracellular Starting from the cDNA fragment of our first library
matrix. Some examples of these genes are listed in Ta- we performed a multi-tissue Northern blotting. Ac-
bles 3 and 4. cording to the expression pattern we ask the Ressource
Center of the German Human Genome Project to pro-
vide a full lenght cDNA clone of the gene by screen-
5.2. Characterization of the human Calcyclin binding
ing one such library of an organ in which the gene is
protein
strongly expressed. In the meantime we ordered the ge-
nomic PAC clone from the Sanger Centre in the UK
We meanwhile fully characterized one gene on chro- which performed the genomic sequencing for FISH ex-
mosome 1q24–q25 [22]. This was greatly facilitated by periments. After confirming and sequencing of the full
the fact that a sequenced genomic clone was available. length cDNA clone is was fairly easy to reveal the ge-
Since the Human Genome Project is finishing its com- nomic structure of the gene by comparing the cDNA
pletion, the entire sequence of the human genome will and the genomic sequence. The gene encodes for the
120 I. Petersen and S. Petersen / Lung cancer classification

human homologue of the Calcyclin-binding protein. It Acknowledgements

was mapped to the 1q24–q25 and spans about 10 kb
of genomic DNA with a 1.5 kb mature transcript. The We would like to thank all the technicians, postdoc-
putative protein encoded for 228 amino acids and har- toral fellows and medical students that contributed to
bors a nuclear localization signal and several protein this work, i.e., Nicole Deutschmann, Manuela Pacyna-
kinase phosphorylation sites. The gene showed over- Gengelbach, Christa Schütze, Jacqueline Rudolf, Cor-
expression in the majority of lung cancer cell lines as dula Heckert, Günter Wolf, Karsten Schlüns, Karl
well as advanced primary tumors. In addition, FISH Roth, Holger Langreck, Glen Kristiansen, Almut Goe-
analysis with a PAC clone at 1q24–q25 covering the ze, Sven Schmid, Blend Krebber, Marco Aninat Meyer.
genomic sequence indicated intrachromosomal and in- We are deeply indebted to our institute director Man-
terchromosomal rearrangements associated with gene fred Dietel for his long term continuous support. We
amplification. The data suggest that the human calcy-
enjoyed the stimulating collaborations with several
clin binding protein acts as a putative proto-oncogene
colleague within and outside the Charité, in partic-
during progression of lung cancer and possibly other
ular Ulrike Bockmühl, Klaus Gellert, Thomas Ried
advanced tumor types. This data was accumulated in a
and Michael Speicher. The work was supported by
couple of months in contrast to years needed in before
all the above mentioned facilities were available. We the Charité university hospital, Deutsche Forschungs-
are meanwhile in the process of generation antibodies gemeinschaft, Berliner Krebsgesellschaft, Deutsche
against the gene to test it more easily on clinical tumor Krebshilfe and the Monika Kutzner-Stiftung.
samples.

References
6. Perspective
[1] M. Bentz, A. Plesch, S. Stilgenbauer, H. Dohner and P. Lichter,
The most important task in the forthcoming years Minimal sizes of deletions detected by comparative genomic
will be first the identification of all genes involved in hybridization, Genes Chromosomes Cancer 21 (1998), 172–
lung tumorigenesis. Out of this pool of probably sev- 175.
eral thousand genes those need to be selected that carry [2] U. Bockmühl, K. Schlüns, I. Kuchler, S. Petersen and I. Pe-
the highest clinical significance. This will require the tersen, Genetic imbalances with impact on survival in head and
neck cancer patients, Am. J. Pathol. 157 (2000), 369–375.
use of modern screening technologies like cDNA ar-
[3] O. Brison, Gene amplification and tumor progression, Biochim.
rays and also sophisticated bioinformatics to analyse
Biophys. Acta 1155 (1993), 25–41.
the wealth of data. These genes need to be assessed on
[4] L. Diatchenko, Y.F. Lau, A.P. Campbell, A. Chenchik, F. Mo-
clinical specimens for which the use of tissue arrays qadam, B. Huang, S. Lukyanov, K. Lukyanov, N. Gurskaya,
provide a very promising tool [7]. Finally the structure E.D. Sverdlov and P.D. Siebert, Suppression subtractive hy-
and function of these genes need to be clarified for po- bridization: a method for generating differentially regulated or
tential therapeutic intervention. Regarding tumour di- tissue-specific cDNA probes and libraries, Proc. Natl. Acad.
agnostics the methodology how these new markers will Sci. USA 93 (1996), 6025–6030.
be investigated in a routine setting will largely depend [5] A. Kallioniemi, O.-P. Kallioniemi, D. Sudar, D. Rutovitz,
on the number of genes that need to be surveyed for J.W. Gray, F. Waldman and D. Pinkel, Comparative genomic
hybridization for molecular cytogenetic analysis of solid tu-
a genetic classification. If a large number of genes, mors, Science 258 (1992), 818–821.
e.g., more than 50, have to be assessed simultaneously,
[6] M.J. Kelley, K. Nakagawa, S.M. Steinberg, J.L. Mulshine,
biomolecules extracted from the tumour tissue proba- A. Kamb and B.E. Johnson, Differential inactivation of
bly will be analysed by chip technologies. This, how- CDKN2 and Rb protein in non-small-cell and small-cell lung
ever, will require a change in classical procedure of tis- cancer cell lines, J. Natl. Cancer Inst. 87 (1995), 756–761.
sue processing in pathology which needs to be comple- [7] J. Kononen, L. Bubendorf, A. Kallioniemi, M. Barlund, P.
mented by cryopreservation since most of these tech- Schraml, S. Leighton, J. Torhorst, M.J. Mihatsch, G. Sauter
nologies require fresh frozen material. However, it is and O.P. Kallioniemi, Tissue microarrays for high-throughput
well perceivable that the number of markers remain so molecular profiling of tumor specimens, Nat. Med. 4 (1998),
844–847.
small that they might still be analysed by conventional
[8] G. Kristiansen, S. Petersen, O. Kaufmann, K. Schlüns, M. Di-
immunohistochemistry one by one. Anyhow, these are etel and I. Petersen, Overexpression of c-erbB2 protein cor-
very exiting years for tumour diagnostics and pathol- relates with disease-stage and chromosomal gain at the c-
ogy is the medical discipline which will provide major erbB2 locus in non-small cell lung cancer, European J. Cancer
impacts and will first benefit from this development. (2000), in press.
 I. Petersen and S. Petersen / Lung cancer classification 121

[9] M.E. Mattson, E.S. Pollack and J.W. Cullen, What are the odds [24] J.R. Pollack, C.M. Perou, A.A. Alizadeh, M.B. Eisen, A.
that smoking will kill you?, Am. J. Public Health 77 (1987), Pergamenschikov, C.F. Williams, S.S. Jeffrey, D. Botstein and
425–431. P.O. Brown, Genome-wide analysis of DNA copy-number
[10] K.M. Müller and S. Gonzalez, Preneoplasias and early carci- changes using cDNA microarrays, Nat. Genet. 23 (1999), 41–
noma of the lungs – histogenetic aspects of bronchial carci- 46.
noma, Pneumologie 45 (1991), 971–976. [25] M.B. Reichel, H. Ohgaki, I. Petersen and P. Kleihues, p53 mu-
[11] K.M. Müller and A. Fisseler-Eckhoff, What’s new in lung tu- tations in primary human lung tumors and their metastases,
mor heterogeneity?, Path. Res. Pract. 184 (1989), 108–115. Molecular Carcinogenesis 9 (1994), 105–109.
[26] T. Ried, I. Petersen, H. Holtgreve-Grez, M.R. Speicher, E.
[12] I. Petersen, A. Schwendel, U. Bockmühl and M. Dietel, Die
Schröck, S. du Manoir and T. Cremer, Mapping of multiple
Komparative Genomische Hybridisierung – eine Screening-
DNA gains and losses in primary small cell lung carcinomas
methode in der genetischen Tumordiagnostik, Pathologe 17
by comparative genomic hybridization, Cancer Res. 54 (1994),
(1996), 333–341.
1801–1806.
[13] I. Petersen, H. Langreck, G. Wolf, A. Schwendel, R. Psille, [27] K. Roth, G. Wolf, M. Dietel and I. Petersen, Image analysis
P. Vogt, M.B. Reichel, T. Ried and M. Dietel, Small cell lung for comparative genomic hybridization based on a karyotyping
cancer is characterized by a high incidence of deletions on program for Windows, Anal. Quant. Cytol. Histol. 19 (1997),
chromosomes 3p, 4q, 5q, 10q, 13q and 17p, Br. J. Cancer 75 461–474.
(1997), 79–86. [28] Y. Sameshima, Y. Matsuno, S. Hirohashi, Y. Shimosato, H. Mi-
[14] I. Petersen, M. Bujard, S. Petersen, G. Wolf, A. Goeze, A. zoguchi, T. Sugimura, M. Terada and J. Yokota, Alterations of
Schwendel, H. Langreck, K. Gellert, M. Reichel, K. Just, S. du the p53 gene are common and critical events for the mainte-
Manoir, T. Cremer, M. Dietel and T. Ried, Patterns of chromo- nance of malignant phenotypes in small-cell lung carcinoma,
somal imbalances in adenocarcinoma and squamous cell carci- Oncogene 7 (1992), 451–457.
noma of the lung, Cancer Res. 57 (1997), 2331–2335. [29] A. Schwendel, H. Langreck, M. Reichel, E. Schröck, T. Ried,
[15] S. Petersen, G. Wolf, U. Bockmühl, K. Gellert, M. Dietel and M. Dietel and I. Petersen, Primary small cell lung carcinomas
I. Petersen, Deletions on chromosome 10q in human lung can- and their metastases are characterized by a recurrent pattern
cer: Association with tumor progression and metastatic pheno- of genetic alterations, Int. J. Cancer (Pred. Oncol.) 74 (1997),
type, Br. J. Cancer 75 (1998), 79–86. 86–93.
[16] S. Petersen, J. Rudolf, U. Bockmühl, K. Gellert, G. Wolf, [30] S. Solinas-Toldo, S. Lampel, S. Stilgenbauer, J. Nickolenko,
M. Dietel and I. Petersen, Distinct regions of allelic imbalance A. Benner, H. Dohner, T. Cremer and P. Lichter, Matrix-based
on chromosome 10q22-q26 in squamous cell carcinomas of the comparative genomic hybridization: biochips to screen for ge-
lung, Oncogene 17 (1998), 449–454. nomic imbalances, Genes Chromosomes Cancer 20 (1997),
399–407.
[17] S. Petersen, G. Wolf, U. Bockmühl, K. Gellert, M. Dietel and
[31] M.R. Speicher, S. Petersen, S. Uhrig, I. Jentsch and I. Petersen,
I. Petersen, Allelic loss on chromosome 10q in human lung
Analysis of karyotypic instability in non-small cell lung cancer
cancer: association with tumor progression and metastatic phe-
by multiplex-FISH (M-FISH) and comparative genomic hy-
notype, Br. J. Cancer 77 (1998), 270–276.
bridization (CGH), Lab. Invest. 80 (2000), 1031–1041.
[18] S. Petersen, M. Aninat-Meyer, K. Schlüns, K. Gellert, M. Di- [32] W.D. Travis, T.V. Colby, B. Corrin, Y. Shimosato and E. Bram-
etel and I. Petersen, Chromosomal alterations in the clonal evo- billa, WHO International Histological Classification of Tu-
lution to the metastatic stage of squamous cell carcinomas of mours: Histological Typing of Lung and Pleural Tumours,
the lung, Br. J. Cancer 82 (2000), 65–73. Springer-Verlag, Heidelberg, 1999.
[19] I. Petersen, H. Hidalgo, S. Petersen, M. Pacyna-Gengelbach, [33] R. Ullmann, A. Schwendel, H. Klemen, G. Wolf, I. Petersen
K. Schlüns, A. Goeze, B. Krebber, J. Szymas and A. von Deim- and H.H. Popper, Unbalanced chromosomal aberrations in neu-
ling, Chromosomal imbalances in brain metastases of solid tu- roendocrine lung tumors as detected by comparative genomic
mors, Brain Pathol. 10 (2000), 395–401. hybridization, Hum. Pathol. 29 (1998), 1145–1159.
[20] S. Petersen, J. Rudolf, U. Bockmühl, N. Deutschmann, M. Di- [34] A. von Deimling, D.N. Louis, K. von Ammon, I. Petersen,
etel and I. Petersen, Analysis of the DMBT1 gene in carcino- T. Hoell, R.Y. Chung, R.L. Martuza, D.A. Schoenfeld, M.G.
mas of the respiratory tract, Int. J. Cancer 88 (2000), 71–76. Yasargil and O.D. Wiestler, Association of epidermal growth
[21] S. Petersen, C. Heckert, J. Rudolf, K. Schlüns, O.I. Tchernitsa, factor receptor gene amplification with loss of chromosome 10
R. Schäfer and I. Petersen, Gene expression profiling of ad- in human glioblastoma multiforme, J. Neurosurg. 77 (1992),
vanced lung cancer, Int. J. Cancer 86 (2000), 512–517. 295–301.
[35] J. Whang-Peng, C.S. Kao-Shan, E.C. Lee, P.A. Bunn, D.N.
[22] S. Petersen, N. Deutschmann, C. Sers, M. Dietel and I. Pe-
Carney, A.F. Gazdar and J.D. Minna, Specific chromosome
tersen, Amplification and overexpression of the gene encoding
defect associated with human small-cell lung cancer; deletion
the human calcyclin binding protein on chromosome 1q24–q25
3p(14–23), Science 215 (1982), 181–182.
in advanced lung carcinomas (2000), manuscript submitted.
[36] W. Wick, I. Petersen, R.K. Schmutzler, B. Wolfarth, D.
[23] C.M. Perou, S.S. Jeffrey, M. van de Rijn, C.A. Rees, Lenartz, E. Bierhoff, J. Hümmerich, D.J. Müller, A.P. Stangl,
M.B. Eisen, D.T. Ross, A. Pergamenschikov, C.F. Williams, J. Schramm, O.D. Wiestler and A. von Deimling, Evidence for
S.X. Zhu, J.C. Lee, D. Lashkari, D. Shalon, P.O. Brown and a novel tumor suppressor gene on chromosome 15 associated
D. Botstein, Distinctive gene expression patterns in human with progression to a metastatic stage in breast cancer, Onco-
mammary epithelial cells and breast cancers, Proc. Natl. Acad. gene 12 (1996), 973–978.
Sci. USA 96 (1999), 9212–9217.
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