JOURNAL
OF CELLULAR PHYSIOLOGY 203:1-5 (2001:
REVIEW
ARTICLE
Early
Diagnosis of Lung Cancer by Detection of Tumor Liberated Protein
GIULIO
TARRO,* ANTONIO PERNA, ANO CIRO ESPOSITO Department of Diagnostics,
Unit of Mo/ecu/ar Bi%gy, "D. Cotugno" Hospita/,
Naples, and Unit of Path%gy "M. Scarlato"
Hospita/, Scafati (SA), /ta/y
Tumor
liberated protein (TLP) is a protein that can be used to reveal
the early development of a tumor. Besides being formed in thl
tumor, TLP is released in the blood when a patient starts
producing cancer cells, which in tum enables the physician to
intervene at, stage when the cancer is operable. To date, the
available studies of tumor markers in lung cancer patients are
CEA, NSE, TPA Chromogranine, CA 125, CA 19-9, and Cyfra 21-1. The
sensitivity and specificity for serum markers ranges between 50 and
90% depending on the study and the cl inical samples analyzed.
Most of these markers show an increased rate of positivity as the
stagl advances. There are very limited data on TLP to draw any
firm conclusion regardingthe diagnostic value ofthis marker. TLP
has beer detected in 53.1 % of non-small celllung cancer (NSCLC)
patients (N = 534) with 75% being positive in the early stage (stage
I) an< dropping to 45% in the late stage (stage IV). However, 7.6%
blood donar sera and 17.4% chronic lung disease sera have also
teste< positive. In a confirmation study, the specificity was 89.94%
and the sensibility was 63.63% from stage 111 to IV NSCLC
patients.ln al initial study ofTLP as a marker for early
detection in stage I, NSCLC patients showed a sensitivity of 66.7%
and a specificity of 800;' for TLP compared to a
sensitivity of 33.3% far CA 19-9, 11.1 % for Cyfra 21-1 and CA 125,
and 0% for CEA; the specificity for ali fou of the latter markers
was 100%. Using immunohistochemical analysis with peroxidase anti-peroxidase
(PAP), we observed tha NSCLC cells were positive; we used the
specific rabbit antiserum to TLP, which turned out negative in
the presence of 1 mg/mlofthl synthetized peptide. The pre-serum
was a/so negative. The same reactivity was found early in the
modified epithelial cel/s o interstitial lung fibrosis and might
be a predictive marker of celi transformation. The site of the
peroxidase positivity wa~ cytoplasmic, of diffuse and/or granular
type. J. CelI. Physiol. 203: 1-5,2005. © 2004 Wiley-Liss, Inc.
EARLY
DETECTION AND RESEARCH STRATEGY IN LUNG CANCER
Lung
cancer is the main cause of mortality in the world for men
between 35 and 70 years of age. The highest incidence rates are
found in Europe and North America. It is estimated that there
were approximately 375,000 cases oflung cancer in Europe in 2000,303,000
in men and 72,000 in women; the resulting deaths totalled about
347,000 (280,000 men and 67,000 women) (Tyczynski et al., 2003).
It is clear that lung cancer is a very important health problem
for men, considering the mortality rate associated with the
disease and the fact that the choices oftherapy for this tumor
are limited. Air pollution, polynuclear aromatic hydrocarbons
mostly derived from car traffic, radon and tobacco smoking surely
play a fundamental role in causing this disease and therefore the
first approach must be prevention measures. However, even though
tobacco smoking and environmental factors' are certain causes of
risk, they alone do not soom sufficient to justify the
differences found in mortality and incidence for lung cancer
within a country and between various countries. One must consider
other important risk factors that act as independent factors or
as modifiers or modulators of the effects of smoking. For this
reason, new approaches are required to counteract this particular
epidemie.
The
discovery of biomarkers, that is markers of the biological
process associated with cell growth, represents a basic target
in tumor diagnosis, sincethey can be used for early diagnosis,
tumor relapse and metastasis, as well as for prognosis, site of
tumor, and rnonitoring of therapy.
Several
studies have shown that the expression of different biornarkers
is frequent in eelliines oflung cancer. and many authors have
oonsidered the presence of these markers in blood as an index of
the extension of disease, prognosis, and l'espanse to therapy.
Some turnor markers
©
2004 WILEY-LISS, INe.
CNSE,
Chromogranine, CEA, TP A, and CYFRA 21-1) havE been evaluated in
many patients with SUlall celI lunli cancer (SCLC) as well as
with NSCLC . ;However, tht diagnosis and follow-up of this
patho'ogy are not ye1 satisfactory, although a relationsmp \Vitti.
clinical relevance has been found for the NSE and chromograninE
markers in SCLC; the CYFRA 21-1 marker, on the othel hand, has
been useful only in the follow-up of NSCLC fOI the squamous
subtype CSoichiro et al., 2001). Recently, there has been renewed
interest in screening as spiraJ computerized tomography can
detect smalI asymptomatic lesions more etfectively than
conventional radiography. Although cure rates for such lesions
are very good, there il; to date no evidence for the
etfectiveness of mass screenin{i strategies (Tyczynski et al.,
2003).
TUMOR
ASSOCIATED ANTIGENS IN HUMAN LUNGCANCER
In
1983 a tumor associated antigen (TAA) was isolated from non small
cell lung (NSCL) cancer (Tarro et al.: 1983) and named tumor
liberated protein CTLP) (Tarro 2000). TLP is composed of soluble
lipoglycoproteins; itf: size measures between 48.1 and 61 A and
it has aIl isoelectric point less than 7.0 and a molecular weight
01 214,000 Da. It was first analyzed for amino acid composition
and then some partial sequences were
Contract
grant sponsor: Foundation T. and L. de Beaumont Bonelli (for
Cancer Research).
*Correspondence
00: Giulio Tarro, Division of Virology, D. Cotugno Hospital, Via
Posillipo n. 286,80123 Naples, Italy. E-mail: gitarro@tin.ìt
Received
6 February 2004; Accepted 29 June 2004 Published online in Wiley
InterScience (www.interscience.wiley.com.J. 25 August 2004.
DO!: 1O.lO02,jcp.20195
2
TARRO
ETAL.
obtained.
A 100 kDa (CSH-275) fragment was identified in TLP and one
epitope was used as an antigen to produce antibodies directed
towards specific sites ofTLP by inoculation in rabbits (Tarro et
al., 1993). This antiserum was named CSH-419 and used by some
authors for research on TAA in lung and colorectal cancer
patients (Garaci et al., 1996; Guadagni et al., 2000). A
reasonably high level of purification of TLP enabled us to obtain
an initial amino acid sequence which in turn led to the synthesis
of the foliowing three specific peptides from lung and colon
cancer (Tarro, 1999): ArgThrAsnLysGluAlaSerlle;
GlySerAlaXpheThrAsn; AsnGlnArgAsnArgAsp. A fourth peptide,
GlyProProGlu ValGlnAsnAlaAsn, was obtained from TLP which was
purified from an urogenital tumor.
Recent
studies with rabbit antibody raised against a peptide from lung
and colon cancer, made up of eight amino acids, revealed that TLP
is produced in the cytoplasm ofthe malignant cells (Table 1, Fig.
lA). Since it was not detected in normal tissues, we concluded
that it was a tumor-specific antigen (Tarro et al., 1998). Our
work on TLP indicates that this protein appears to be a new tumor
marker with potential clinical applications. Using Western blot
analysis, we were able to show that TLP was expressed in alilung
cancer-derived celilines, in breast cancer-derived celi lines,
and in colorectalderived celilines that were studied (Tarro et
al., 1998). Since lung and intestinal tissues have a common
embryonic origin, it is not unreasonable to suggest that TLP
might be tumor-specific antigen for at least some of the
malignancies that arise from tissues of epithelial origino Since
we were not able to detect TLP in normal lung tissues, nor in the
osteosarcoma celiline SAOS-2 (Tarro et al., 1998), the notion
that TLP is indeed a tumor-specific antigen is supported. On the
basis of the immunohistochemical studies which revealed a large
amount of TLP in the columnar epithelial celis and lumen (secreted
TLP), as illustrated in Figure lA, it appears that TLP is a
cytoplasmic antigen which is specific for epithelial celi-derived
neoplasms. Preincubation ofthe antibody with the corresponding
immunizing antigenblocked the immunocytochemical reaction, thus
confirming the specificity of the antibody (Tarro et al., 1998).
Immunohistochemical
evaluation
of
the role of TLP
lmmunohistochemically,
all the lung cancer specimens with high or medium levels of
difIerentiation
showed
TLP cytoplasmic immunostaining (Table 1). The evaluated
colorectal carcinomas also were positive for TLP expression (Tarro
et al., 1999). No TLP protein was detected in the surrounding
normal tissues or in inflammatory celis. In some specimens tms
specific staining was also recognized, as well as in the lumen of
atypical glands and in bronchial secretion. This last observation,
taken together with TLP expression in all the adenocarcinoma-derived
celllines (including MCF-7 and HT-29) and tumor specimens, could
suggest that TLP is a secretory product ofthe neoplastic cells.
On the other hand, the lower differentiated squamous carcinomas,
the small celliung carcinomas (well known to be an aggressive
type oflung cancer) and the two sarcomas did not exhibit any TLP
immunostaining (Tarro et al., 1999).
Combining
the immunohistochemical data with the results of the Western blot
analyses, we conclude that TLP is a cytoplasmatic antigen
specific for epithelial celi-derived neoplasms only, because it
was not detected in non-neoplastic tissues celis, in
neuroendocrine tumors, or in mesenchymal cell-derived tumors and
normal cells (Rasi et al., 2000). In addition, finding that TLP
is detectable in neoplasms and tumor celilines with common
embryological features seems to suggest that TLP might be an
"oncofetal antigen."
IMMUNOISTOCHEMICAL
TEST BY PEROXIDASE
ANTI-PEROXIDASE
(PAP)
Immunoreactivity
for TLP was found in neoplastic tissues by anti NQRNRD serum
prepared after rabbit immunization obtained by using the sequence
length of six amino acida (Tarro, 1999) according to the pattern
already perforrned in the Cold Spnngs Hs.rbor Labora-
tory (Tarro et al., 1993). y
The
immunostaining was always ~wasmic, with a low- to-absent
background. AlI adenocarcmomas showed a high expression level
ofTLP (Fig. lC,D). Tbe protein was distributed either in small
granules or in larger accumulations (Fig. 2A.B); in some
specimens, TLP was detected in the lumen of atypical glands and
in the bronchial secretions (Fig. 2C,D), suggesting that TLP
could be considered a secretory product ofthe neoplastic cells.
We found cytoplasmic-specific TLP staining of various expression
levels in alI medium- and welldifferentiated specimens of
squamous carcinomas (Fig. lE). However, TLP was noi detected in
the infiltrating lobular carcinoma of the breast (Fig. 3e), urothelial
carcinoma (Fig. 30), or in the myocardium
TABLE
1. Reactivity of rabbit antiserum to the TLP-derived peptide (RTNKEASIC)
Organ
l.Lung
2.
Lung
3.
Lung
4.
Lung
5.
Lung
6.Lung
7. Lung 8.Lung
7.
Lung
8.
Lung
9.
Lung
10.
Skin
11.
Breast
12.
Breast
13.
Thymus
14.
Endometrium
15.
Bladder
16.
Colon
Origin
of tissue
Normal
adult
Fetus
18 weeks
Interstitial
inftammation Bronchiolo-alveolar adenocarcinoma Acinar
adenocarcinoma
Papillary
adenocarcinoma
Epidennoid
carcinoma (differentiated) Epidermoid carcinoma (non-keratinizing)
Small celi carcinoma
Sarcoma
Malignant
mesothelioma Malignant melanoma Infiltrating duct carcinoma
Intraductal adenocarcinoma Malignant thymoma Adenocarcinoma
Papillary
urothelial corcinoma Adenocarcinoma
Peroxidase
positivity
o
O
O
3+ 3+ 3+ 2+ o o o O O
O
O
o o
O
2+
!,:,\RLY
llETE<.'TIOt': ClF lXt':(; C,\:\('I-:H IJY TU'
17.
Fig.
I. Histological seclion or n lung udennc(lI'cillomu incubated
wilh Uw rabbit antiscrum to thc tumor liberated prlliein ('l'LP) pcptidc
I{TNKEi\SI (A) und or CI squumous lung "arcinomu IDJ
"" or u lung ndenoclll'cinoll1u \e anel DI incubull'd
wii h the rahbit ant.iserulllln t.hc 'l'LP pcpt ide NC-tHNRD. The
bruwn slaining n'p,.('s('nL~ the dii't.riblltinn nfTLP in t.hcsc
malignanlli~SlWi' lA. B and D: ,·180: C: . R~()I.
lTable
21. A rnild oxpression level 01' l'LP was a]so dctccted in thc
colorccLal adenocarcinoma examined cFig. 3A). Some outhors
detected TLP in 53.117, 01' NSCL cancer patients, with 07r
positivity in paticnts with tumors other than NSCL cancer;;, 7.6'ì;
positi\'ily in unknown blood donors, alld 17.417, positì"ity
in patients with chronic lung discascs h:wing ;)11 elevated risk
far lung cancer (Garaci et al, 1996). 'l'ho antibody to NQRNRD
sequence stains the alveolnr epithelium modified in interstitial
lung fibrosis (Fig. 3B), 'l'his epithel ium changes from Oai io
cubical: ihis alteration is tel'll1cd cubica] allomol'ph iSI11,
indicating a retu rn of the alveolar epitheliul11 to the look of
thc retallung berorc cxhibiting respil'aiory functions. Lung
parcnchyma with diffuse inten;titial fibrosis does noi clisplay
uny respiratory funciion due to thc compleie vanishing 01' septal
capillaz'y circulation in this pathoJogy, as in t.h fetal lung {Perna
et al., 1999L The pm'enchYl11a with
uch
Inl)(JjfJcatiuIlS is Illore exposecl io neoplastic iransfOl'll1ation
(Garaci et al.. 1996), Thc study of thc c1istribution 01' anti-TLP
anlibodies in heallhy pcople a Il cl patients wilh neoplasia, or
in other patients afTcctcd by Jung inflammator)' paiholog-ies who
could l'alI L1nder thc category or Iling ncopla:o:i'l, is very
interestin,g IEsposiio et al .. 1997), 1/1 ract, lhe rcuctivil)'
Si),,'11UI in genel'8 I pat.hologie:; i!'l n uflcfiJ J tcst to USSCSi:ì
thc spccilìcity anel posit.ivc prcdict.ive v\llua 01' Lh\;! atil;iay.
Finally,
rigo
2. TLP iIllIl111nohisluehclllll',,1 cxprcssiull b," thc
alllibud~' lu NqR:--lHD ~eqtll'nrl'. A l'rpl'Psl'nIlllil'1' ~taining
in a lungo ndplloral'eiIwnHl \A: . :i20: B: . 4S0: c: . :320: D:
. -ISO I.
we
believe that scnsltlvlty can increase scnsibility. with il11provement.
o[ thc lst generaiion test (Tarru unel Esposito. 200~1.
POLYCLONAL
ANTLBODY AND "ELI SA SANDWICH"
Previou:>
siudies have shown thc l'cadivity or the 1'I.lbbit anLiserlllll
ubtainccl againsi lhc pcptide seqllencc RTNKEASl ('l'alTo el al
.. ] 993. ] 998. :2001: Garaci eL al.. ] 996: 'l'ano, 1999, :2000.
2002: Rasi cl al.. 2000),
inl;e
this scquencc is analogous to that present in cOl'in (RTQKEASIl, :J.
proteic companent or h1ll1lan myocarclilll11. polyclana]
antibodies were procluced b,v PRIMM (Milan. lLaly) in rabbiis
againsi boih varianis l and 2 or I;orin w,l.. l'or lbc scqllcnl;c~
of junciìon or delction, respe<.:tively, to c1eten11ine
whether the 'l'LP \Vas homologous or onl." 311alogolls lo lh!'
COl'in.
An
ELlSA sandwich was preparcd using t.hc rabbit aniiserum allii-RTNKEASI-KLH
as a capillring aniibody. purifìcd for immune affìnit.v (l;.\l
with pcpticle. whel'c:ls c1irrerent antisera were usecl as t.he
tracing' anLibody: poly Ab anti val' l delclion-BSA pl1l'i/ied
wit.h proiein A: pol.v anti val' ] dcletion-KLH pllrifìccl l'or
lA wilh pcpLide; p01v anlì \'tlr l juncl.ion-BSA plll'ificci
wiih pl'olein A: pul." anii valI jllnction-KLll purifll.:d l'or
lA wit.h pt'pticle: poi} 'lnti V\.1l' 2 deletion-BSA punllcd wlth
proicin A; poJy anli val' 2 dclciion-KLH
4
|
TARRO
ET AL. |
18.
sera
were firsL purified against. pepLide + BSA and then after
gel filtration on a NAP 25 column tpharmacia, Uppsala,
Sweden l, as required by the conjugation proLocol (NaHCO;l
0,1 M -l- 0.9'H NaCI). Artel' gel filtration,
lyophilization, and dissolution, the purified sera had
higher antibody titcrs. The serum + EZ Iink was tested at
diITerent dilutions (1:10,1:50, 1:100, 1:1,000) and a
significantlossofactivity was measul'ed, When w Lested
the sera at lower di lutions (1:10, 1:50, l: 100l, the
results confirmed the loss or adivity. The
rcsuspension of Iyophilized sel'um in water doe not make
it possible to obtain a homogeneous solution, but l'ather
one conLaining precipitates, Therefore, the conjugation
on 0,3 mg in 50 ~d or total and non-specilic IgG,
conducted according to the proLocol, might have
limitations l'or thc final yield, FurLhermore, we
evaluated the immunohistochemical expl'ession ol'TLP
using the polyclonal antibodies to corin w,t, val'iants 1
and 2 l'or thc sequence ofjunction ancl deleLion, TLP was
not dctccted in the clilferenLiatccl adenocarcinomns by
ali the rabbit antisel'a (Fig. 4A,BJ, whereas the
immunostaining was low to absent in the myocardium (Fig,
4C,Dl, AIMS
AND PERSPECTIVES FOR TLP Ongoing studies have
attempted Lo achieve the complete seguence 01' TLP (Tano,
2002), which in turn |
Fig.
3. 'l'LP imlllulluhistochcmicai cxpression hy t1w antibòdy
lo NQRNRD sequence, A reprcscnl:ui\'c staining in a colo-l'celaI
carcinomn tA:'480), Thc anlibod.\' LO NQR~RD scqucncù
slaim; thc alvenlar L'pithcliull1 Illodificd in thc
inlerstitiallung fibrosis (B: ,4801, NCI!Hlivc l'n!' TI.P
illll1lllnostainiog nn adcnocal'cinomfl or lhc hrcast le:
,480. am} an urulclial curcinol1llJ IO: . :l201, |
purifìcd
for lA with pcptide; poJy anti val' 2 junctionBSA
purified with protein A: poly anti var 2 junctionKLH pl.lrifìed
for IA with peptide. Ali
antibodies were pUl"i{-jed by the poDI or
antiscra of two l'abbits illlmunizeci and conjugated with
biotine or with horseradish peroxidase (HRPl. Amigen
samples were human myocardiulll anel norl1131 lung, The
cuncenLl'atinn 01' HeLa cells and NSCLC extracts were
lanci 511g/ml. respcctively, As Lhe control, an ELlSA
standarel plaLe wa:; pl'cpal'ed to check thc con'cct
procedure for the use uftbe Lestccl antibodies (gel
fìltl'ation. Iypbilization, and dissolution in HzO,
conjugaLion); thc coating wa pCl'fol'mecl with tbc
antigens by which tbe antibodie against thc corin
variants were induced in rabbits, The |
TABLE
2, Hcactivil.v or ,'abhit antiscl'lIm LO thc TLP-dcrived
peplidc fxQRNHlJl |
rgun
|
PCl'Oxidnsc
posi! h'il,\" |
19.
l\ol'muluduil
I
nle!'slili,,1 libl'O;is ,\dcJJuciI n.:illlJrna Epidermoid
clrdnuma M\'OCflrciiuITI 1.;fillrHling
dUCI cnrcinurrw .-\dl'nOCOI'cinOIlI!t C
,..,theltul Cill.,.inoll1o |
o
|
;1.
~ . n l) |
I)
|
fig.
'I, A Iling ~.dcnOC1II'cllloma nl'gnti\'(; liJI' 'l'LP iml11unOSlllining-
ily thL' :lIllibud)' lo tlH.' cOl'in v:t .
iaol I ,,l' jllnctioll lA: .4801 and by lhe anlibodv 111 lh"
co!"in \'<1,';al1l 2 or juoclion (B:, ·ISO), A human
1I1.\·uulrcliulll nq;ut hT l'or illlmUIlOt'i1 ni,iillg
by ih~ ;-'nt ihn,.1~· fo t hp
{'m'in \'ill'innll ofjullctiunlC: ,4801 <lncl ~lighUy
pll-"ili\'c fili' imlllunnSloillillg 1;.1 thc>
anlihod,Y lo thc cOl'in variflnt 2 ofjuncliu" IU: .411111.
|
EARLY
DETECTION OF LUNG CANCER BY TLP
5
should
enable in vitro preparations oflarge quantities of TLP by genetic
engineering. The RT-PCR of the TLP fragment (300 bp), from the A549
celi line, was subcloned into the PGEM-T easy plasmid (vector)
(Promega, Madison, Wl), resulting in the T-easy TLP construct (3,300
bp). The fragment (300 bp) could be eleaved with EcoRI. A
putative open reading frame was deduced (Tarro, 2002).
The
availability of such antigen preparations will facilitate future
studies on the role of TLP in human malignancy. It could also
enable the preparation of an assay for early diagnosis of the
corresponding tumors (Soichiro et aL, 2001; Tarro and Esposito,
2002) and might even be useful in the generation of a specific
anticancer vaccine to prevent neoplastic disease in subjects at
risk of developing cancer, through the stimulation of the
immune system by preparing on attack against celis that express
this protein.
With
the aim of identifying with the potential role of TLP in early
lung cancer detection, anti- TLP antibodies were studied in lung
cancer patients (Tarro and Esposito, 2002). Low sensitivity could
result from the appearance of specific immunocomplexes able 10 block
the detection ofTLP, when anti-TLP antibodies appear in the serum
ofthe patient. In view ofthis possibility, we performed a study
to determine the presence of anti- TLP antibodies in sera from
NSCL cancer patients and also 10 determine whether these
antibodies represent an immune response to TLP related to human
lung cancer (Esposito et al., 1997). It was also important to
show the correlation of the antibody with the time of appearance
of the TLP in the blood of the patients (Tarro and Esposito, 2002).
Furthermore, in 1997 it was shown for the first time that this
antigen obtained from human lung cancer is capable of producing a
humoral immune response (Esposito et a!., 1997).
Hybridomas
producing anti-TLP monoclonal antibodies (MAbs) were produced
using the somatic fusion method with spleens of mice immunized
with TLPderived peptide RTNKEASIC (paapep), conjugated with KLH
and BSA (Tarro et al., 2001). Among the MAbs selected, eight
belonged to the IgM elass and two to the IgG class. One of these
MAbs, lF2/11, was selected for the setting up of an ELISA for
titration of TLP in biologica! fluids. The test is based on the
competition of the MAbs for antigen coated on microtiter plates
and antigen in solution. The ELISA is able to recognize not only
pep9aa but, more importantly, a crude preparation ofTLP, TLP in
tumor extracts and TLP in human serum (Tarro et al., 2001).
For
only six lung adenocarcinomas (Perna et al., 2001), we used the
monoclonal antibody (MAb) 10 TLP diluted 1:100 on a single
section with the goal oflooking for a possible relationship
between intracellular TLP concentration, degree ofneoangiogenesis,
and extension of angioinvasion (Ray and Stettler-Stevenson, 1994;
Rak et al., 1995; Fontanini t al., 1997; Pema et al., 1999).
In two cases, we observed a high level of intracellular TLP, a
moderate degree of neoangiogenesis and lack of angioinvasion; in
four cases, a high level ofintracellular TLP corresponded with
extended neoangiogenesis and elear-cut angioinvasion (Pema et
al., 2001).
In
conclusion, the results of our study emphasize the importance
ofneoangiogenesis and angioinvasion in the
evolution
of malignancies capable of strongly influencing survival, as
well as the antigenic proteic products associated with neoplastic
tissue (TLP among them) and their possible role in the process of
metastasis.
N
ew studies presented in this review artiele show that a rabbit
antibody raised against a second peptide of six amino acids (NQRNRD)
from NSCLC belonging to the same protein previously detected and
characterized (TLP) is produced in the cytoplasm of malignant
celis. Our study indicates that this protein appears early in the
modified epithelial cells of interstitiallung fibrosis and might
be a predictive marker of celi transformation. These findings
might allow for the preparation of an ELISA with a first sandwich
between the polyclonal antibody against RTNKEASI and the serum
sample capturing the antigen, and then a second inununological
reaction between the second rabbit antibody (antiNQRNRD) and
the previous complex for revealing the reactivity. A specificity
>95% and an increased sensitivity would yield a good method
for the detection ofTLP antigen in the early stages oflung cancer.
LlTERATURE
CITED
Esposito
c, Tarro G, Cuomo N, Morelli F. 1997. Anti-TLP antibodies in lung
cancer patients. Int Med 5:191-194.
Fontanini
G, Lucchì M, Vignanti S, Mussi A, Ciardiello F, De Laurentis M,
De Placido S, Basolo F, Angeletti CA, Bevilacqua G~1997.
Angiogenesis as a prognostic indicator of survival in non-small
celIlung carcinoma: A prospective study. J Nati Cancer Inst 89:881-886.
Garaci
E, Sinibaldi P, Rasi G. 1996. A new tumor-associated-antigen
ofnon-small celI lung eancer: Tumor liberated protein (TLP) a
possible turnor marker. Antieancer Res 16:2253-2256.
Guadagni
F, Graziano P, Roselli M, Mariotti S, Bemard P, Sinibalsi-Vallebona
P, Rasi G, Garaci E. 2000. Differential expression of a new tumor-associatedantigen
l'LP during human colorects1 cancer tumorigenesis. Am J Pathol
154:993-999.
Perna
A, Tarro G, Esposito C, Baldi C, Annunziata S, Morellf'F, Cuomo N,
Di Spirito A 1999. Neoangiogenesis and prognosis in lung can""'t-
Int Med 7: 111113.
Pema
A, Tarro G, Pema F, Esposito C. 2001. Irnrnunocl\elJUcaI
evaluation of endothelal damage by angioinvasivity. Int J Clin
Invest 9:3'l-33.
Ra.k
JW, St Croix BD, Kerbel RS. 1995. Consequences of angiogenesis
for tumor progression, metastasis and cancer therapy. Anticancer
Drugs 6:3-18.
Rasi
G, Sinibaldi-Vallebona p. Serafino A, Bernard P, Pierimarchi P,
Guorino E, Faticanti-Scucchi L, Graziano P, Guadagni F, Garaci E.
2000. A new human tumor-associated antigen (TLP) is naturally
espressed in rat DHD-K12 colorecta1 tumor cells. Int J Cancer 85:540-544.
Ray
JM, Stettler-Stevenron WG. 1994. The role of matrix
metalloproteases and their inhibitors in tumor invasion,
metastatis and angiogenesis. Eur Resp J 7: 2062-2072.
Soichiro
A, Hidek:i K, Neomichi I, Yasushi N, Masayuk:i S, Michiko A,
Takayuk:i K. 2001. Optimal combination of seven tumor markers in
prediction of advanced stage at first examination of patients
with non-small celI lung caneer. Antieancer Res 21:3085-3092.
Tarro
G. 1999. Tumor liberated protein (TLP). 1ta potential for
diagnosis and therapy. Anticancer Res 19:1755-1758.
Tarro G.
2000. An overvÌew of the lung turnor liberated protein (TLP).
Characterization
or the genetic immunologic profile. Int Med 8:5-8.
Tarro
G. 2002. Characterization of a fragment containing a putative TLP
eDNA sequence. Anticancer Res 22:2693-2696.
Tarro G,
Esposito C. 2002. Progress and new hope in the fight against
cancer:
Novel
developments in early detection oflung cancer. Int Med 10:7-11.
Tarro
G, Pederzini A, Flaminio G. Maturo S. 1983. Human tumor WltigeDS
inducing in vivo delayed hypersensitivity and io vitro mitogenic
activity. Oncology 40:248-254.
Tarro
G. Marshak DR, Peroa A, Esposito C. 1993. Antigenic regioOB of
tumor liberated protein complexes and antibodies agaiost the same.
In: Carpi A, Sagripanti A, Grassi B. editors. Third Internation
Congress. Advances in Management of Malignancies. Pisa, ltaly: 6/10
Decemher 1993. Biomed & Pharmacother 47:237.
Tarro
G, Giordano A, Esposito C, Pema A, Claudio PP. 1998.
ImmunoistochemieaI eharaeterizatioo of turnor liberated
particles (TLPl expression pattern in lung cancer. Anticancer Res
18:2365-2370.
Tarro
G, Perna A, Esposito C. Morelli F, Cuomo N, Di Spirito A 1999.
Airns and perspectives for tumor liberated protein (TLP). Riv It
Rie Med Chir 7:43-46.
Tarro
G, Esposito C. Perna A, Cuomo N, Di Spirito A, Morelli F. Perna F,
Maio A, Nolli ML. 2001. Production and characterization of
anti·TLP derived peptide (RTNKEASIC) monoclonal ant.ibodies. Int
Med 9:55-61.
Tyczynski
JE, Bra)' F, Parkin DM. 2003. Lung canoer in Europe in
2000:
Epidemiology,
prevention, and early detection. Lancet QnCQI 4:45-55.
-