ARTICLE
Auteur(s) :, Charles
Dumontet1, Sylvie Isaac2, Pierre-Jean
Souquet3, Françoise Bejui-Thivolet2, Yves
Pacheco3, Nadine Peloux1, Anthony
Frankfurter4, Richard Luduena5, Maurice
Perol3
1Inserm U453, Service d’hématologie, Centre
hospitalier Lyon-Sud, 69495 Pierre-Bénite Cedex
2Services d’anatomopathologie, Hospices civils de Lyon,
France
3Services de pneumologie, Hospices civils de Lyon,
France
4University of Virginia, Charlottesville
5University of Texas Health Science Center, San Antonio,
USA
Taxanes are active agents in patients with non-small cell lung
cancer. In patients with non-small cell lung cancer treated with
single agent taxanes or regimens combining taxanes with other
compounds such as ifosfamide, cisplatin or gemcitabine, response
rates of 7 to 56% have been reported with median overall survivals
in the range of 7 to 14 months [1-4]. Taxanes are original among
chemotherapeutic agents in that their intracellular target is the
mitotic spindle rather than DNA [5]. In contrast to the vinca
alkaloids, taxanes bind to polymerized tubulin (microtubules) only
[6]. It is believed that the binding site of taxanes on
microtubules involves beta tubulin [7]. Although taxanes have been
shown to increase the microtubule polymer mass of tumor cells in
vitro, its antimitotic action in the clinic is likely to be due to
reduced microtubule dynamics (reviewed in [8]). When considering
the effect of taxanes on their intracellular target, microtubules,
it is thus important to consider both the ability of microtubules
to bind taxanes, and the dynamic behavior of
microtubules.Microtubules are complex polymers consisting of
tubulin dimers (containing one α tubulin and one β tubulin
molecule) and a variety of microtubule-associated proteins (MAPs).
In humans there are a number of tubulin isotypes which mainly
differ in their C-terminal sequence [9]. Although the functional
specificity of tubulin isotypes remains controversial, it has been
shown that some the expression of the tubulin isotypes is
tissue-specific [10]. Few data are currently available regarding
the expression of microtubule components in tumor samples or in
their healthy counterparts.The multidrug resistance phenotype (MDR)
due to overexpression of the P-gp transmembrane protein is the best
described mechanism of resistance to taxanes [6]. Other mechanisms
involving microtubules, the intracellular target of taxanes, are
also likely to be involved in the development of chemoresistant
phenotypes [11]. Various investigators including ourselves have
shown that alterations in microtubules, the intracellular target of
taxanes, are associated with resistance to taxanes in in vitro
models [8, 12, 13]. Alterations in the level of expression or gene
sequence of microtubule components have been reported to be
correlated with sensitivity to taxanes in ovarian cancer samples
[14]. More recently Monzo et al. have reported that mutations in
the class I beta tubulin gene are associated with poor prognosis in
patients receiving taxol for lung cancer [15]. These data suggest
that altered microtubule structure is likely to be a mechanism of
resistance to taxanes in the clinical setting.In this study we
analyzed by immunohistochemistry pretreatment samples of patients
with non-small cell lung cancer prospectively included in clinical
trials with taxane-based regimens. We studied the level of
expression of microtubular components, including β tubulin
isotypes, δ2 α tubulin, tau protein, and Pgp responsible for the
classical multidrug resistant phenotype, and correlated these
biological results with patient outcome.
Material and methods
Patients and samples
The analysis was performed on samples from nineteen patients with
non-small cell lung cancer treated between November 1995 and March
1999 in the Pneumological Departments of the Hospices Civils de
Lyon, France.
Histopathological analysis
Immunohistochemical analyses were performed on paraffin-embedded
sections of pathological samples obtained before therapy. Samples
consisted of bronchial biopsies in 10 cases, lymph nodes in 5 cases
and surgical biopsies in 4 cases. The antibodies used were pan-β
tubulin from Sigma, anti-class I β tubulin isotype (produced by
Richard Luduena), anti-class II (7B9 clone) and class III (TUJ1
clone) β tubulin isotypes (produced by Anthony Frankfurter), anti-
δ2 α tubulin (produced by Didier Job, CEA, Grenoble, France),
anti-tau protein from Euromedex (Strasbourg, France) and anti-Pgp
(classical “MDR”) (JSB1 clone from). Immunohistochemistry was
performed on tissues fixed in Bouin or Bouin-Holland fixative and
embedded in paraffin. Routine 5 μm thick paraffin sections
were stained with haematoxylin-eosin-safran. The
immunohistochemical studies were performed on microwaved paraffin
sections using the standard avidin-biotin peroxidase complex
technique (Duet kit, Dako). The antigen retrieval procedure used
for all antibodies was microwaving in citrate buffer.
Statistical analysis
Complete response was defined as the disappearance of all signs of
disease both at clinical examination and on the CT-scan. Partial
response was defined by a reduction of more than 50% in the sum of
products at the largest perpendicular diameter of all tumor
localizations, with no new tumor lesions. Stable disease was
defined as a variation of tumor lesions by less than 50% decrease
or 25% increase. Tumor progression was defined as an increase in
the size of tumor lesions by more than 25% or the appearance of a
new lesion. Overall survival was calculated as the time between the
beginning of chemotherapy and death or last follow-up.
Progression-free survival was calculated as the time between
beginning of treatment and date of tumor progression. Overall
survival and progression-free survival curves were constructed
according to the method of Kaplan and Meier and comparison of
survival curves were performed using the log-rank method.
Immunostaining parameters were considered as dichotomic variables.
Tumors were considered positive for a given marker when the
percentage of cells expressing the protein in the tumor was greater
than the median value in the entire patient population. All
statistical analyses were performed using Statistica 5.0.
Results
Patient characteristics
Patient characteristics are shown in table 1( Table 1 ). Samples were obtained from nineteen
patients, including 18 males. The median age was 57 years (range
45-65 years). All patients had received a taxane-based regimen as
first-line treatment, including paclitaxel-cisplatin combinations
in 17 patients and single agent docetaxel in 2 patients. The
pathological samples analysed were bronchial biopsies in 10 cases,
involved lymph nodes in 5 cases and surgical lung biopsies in 4
cases. Nine patients (49%) had adenocarcinoma, eight patients (42%)
had squamous cell carcinoma and two patients (11%) had
undifferenciated large cell carcinoma.
Patient outcome is shown in table 2( Table
2 ). Eight patients responded (one complete response and
seven partial responses) yielding an overall response rate of 42%.
The median overall and progression-free survivals of the entire
patient population were 320 and 165 days, respectively, in the
entire patient population and 541 and 368 days, respectively, in
responding patients.
Table 1 Patient characteristics
|
Characteristic
|
Number of patients
|
Percentage of total
|
|
Total
|
19
|
100
|
|
Age median
|
57
|
|
|
range
|
(45-65)
|
|
|
Sex
|
|
Males
|
18
|
95
|
|
Females
|
1
|
5
|
|
Type of lung cancer
|
|
Adenocarcinoma
|
9
|
47
|
|
Squamous cell
|
8
|
42
|
|
Undifferentiated
|
2
|
11
|
|
Pathological sample available
|
|
Bronchial biopsy
|
10
|
53
|
|
Lymph node
|
5
|
26
|
|
Surgical biopsy
|
4
|
21
|
|
Treatment received
|
|
Cisplatin taxol
|
17
|
89
|
|
Single agent docetaxel
|
2
|
11
|
Table 2 Patient outcome
|
Clinical parameter
|
Number of patients
|
%
|
|
Response to taxanes
|
|
Complete response
|
1
|
5
|
|
Partial response
|
7
|
37
|
|
Stable disease
|
4
|
21
|
|
Progressive disease
|
7
|
5
|
|
Overall survival
|
|
median
|
320 days
|
|
|
range
|
116-1150
|
|
|
Freedom from progression
|
|
median
|
165 days
|
|
|
range
|
0-450
|
|
|
Tumor progression
|
|
Yes
|
14
|
74
|
|
No
|
5
|
26
|
|
Death
|
|
Yes
|
17
|
89
|
|
No
|
2
|
11
|
Immunohistochemical data
Results of immunostaining of tumor samples are summarized in table
3( Table 3 ). All tumor samples were
positive with pan β and class I tubulin antibody (data not shown).
A majority of samples were found to express class II and class III
β tubulin (89 and 83%, respectively). Examples of positive samples
are shown in ( figure
1 ). Tau protein was expressed in half of the tumor samples
analysed (( figure
1 )). Delta 2 alpha tubulin, the content of which is
correlated with microtubule stability in vitro, was expressed in
one tumor sample only (( figure 1 )). Pgp expression
was found in one tumor sample (data not shown). The percentage of
cells positive for a given marker in tumor samples, differed
significantly, with 100% of positive cells in all tumors for total
β tubulin and class I β tubulin isotype, and median values of 30%
for τ protein, 40% for δ2 alpha tubulin, 50% for class III β
tubulin and 80% for class II β tubulin (table 3).
Expression of these microtubular components was compared to that
found in non neoplastic lung tissue (( figure 2 )). In normal lung
tissue, all cells were stained with pan-β tubulin antibody. Class I
β tubulin antibody stained the bronchial epithelium, particularly
in the basal area. Class II expression was weak in the epithelium,
bronchial glands, alveoli and strong in myocytes and nerves ((
figure 2 )).
Class III staining was found only in certain cells of the
epithelium (( figure
2 )). Class III expression was negative in alveoli and
strong in nerves (data not shown). Tau protein was not expressed in
the epithelial lining and expression was weak in alveoli (data not
shown). δ2 α tubulin was strongly expressed at the apical pole of
the bronchial epithelium (( figure 2 )).
Table 3 Results of immunohistochemical staining
|
Antibody
|
Number of negative samples
|
Number of positive samples
|
Positive cells in positive samples (%)
|
|
Pan β
|
0 (0%)
|
19 (100%)
|
100
|
|
Class I β tubulin
|
0 (0%)
|
19 (100%)
|
100%
|
|
Class II β tubulin*
|
3 (17%)
|
15 (83%)
|
80% (50-100%)
|
|
Class III β tubulin
|
2 (11%)
|
17 (89%)
|
50% (5-100%)
|
|
Delta 2 α tubulin
|
18 (95%)
|
1 (5%)
|
40%
|
|
Tau protein*
|
9 (50%)
|
9 (50%)
|
30% (5-80%)
|
|
MDR*
|
17 (94%)
|
1 (6%)
|
9%
|
*In some cases the total number of samples was less than
19 due to inavailability of sample or technical problems.
Correlation of MT component expression with patient
outcome
A high level of expression (greater than the median value in the
entire population) of class III β tubulin protein in tumor cells
was correlated with a shorter progression-free survival (median 41
days vs. 288 days in patients with low class III expression, (
figure 3 ), p =
0.02). There were 2 responses among 9 patients (22%) with high
levels of class III tubulin vs. 6 among 10 patients (60%) with low
levels of expression (Fisher exact test: p = 0.11). The median
overall survival was 297 days in patients with high levels of class
III isotype and 402 days in patients with low levels (p = 0.74).
Markers other than class III tubulin isotype content were not
correlated with freedom-from-progression nor with overall survival.
Discussion
Until the recent report by Monzo et al., there were no predictive
tests for chemosensitivity to taxanes in patients with lung cancer
[15]. These authors showed that mutations of class I β tubulin,
which represents the most abundant tubulin isotype, were correlated
with adverse outcome in terms of response and survival. These
authors found mutations in 33% of patients, including major
sequence alterations (frameshifts or premature stop codons) in 6
cases (38%). Most of these mutations concerned the GTP-binding site
rather than the putative taxane-binding site. Howere other authors
have not confirmed the existence of genetic polymorphisms in
tubulin beta isotype I genes, either in normal samples nor in lung
cancers [16]. Immunohistochemical analysis of tubulin isotypes by
Monzo et al. failed to correlate tubulin protein expression with
response or survival but the antibody used to detect class I
antibody in this study also detected class II tubulin, which we
found to be present in 83% of our samples. The present study was
performed with a class I specific antibody, which does not cross
react with purified tubulin isotypes other than class I. The
specificity of the antibody against class I tubulin was further
confirmed by the characteristic staining pattern observed in normal
lung tissue.
Our results suggest that high levels of expression of class III
β tubulin isotype by tumor cells is associated with shorter
progression-free survival, with a trend towards reduced response
rates. Class III β tubulin was expressed by a large majority of the
tumors tested, but with a wide range of positive cells in different
tumors (5-100%). The expression of other tubulin isotypes and tau
protein was not correlated with response or survival. Class I β
tubulin isotype was expressed in 100% of cells of all tumors. Class
III β tubulin isotype has been reported to be overexpressed in
various models of cell lines resistant to tubulin-binding agents,
including paclitaxel and vinblastine [13, 17]. The classical
multidrug resistance phenotype mediated by overexpression of the
170 kd Pgp protein is a well described mechanism of resistance to
taxanes in vitro. However expression of Pgp by tumor cells of
patients bearing NSCLC appears to be unfrequent [18, 19]. In our
study sample Pgp expression was detected in one patient only
(6%).
It is not yet clear by what mechanisms the alteration in
microtubule components can alter sensitivity to antitubulin agents.
The two main hypotheses are that there may be alterations in the
taxane binding site on the tubulin dimer [20] and that the
microtubules contained in the tumor cells have different dynamic
properties and may thus be less sensitive to taxanes [21, 22]. It
has been shown that tubulin isotypes, in particular class III β
tubulin isotype, have different dynamic properties. It has also
been shown, in a study comparing the dynamic properties of
microtubules composed of either αβII, αβ III
or αβIV dimers, that the dynamics of microtubules made
of αβIII dimers were the least sensitive to paclitaxel
[23]. It is therefore possible that alterations in tubulin isotype
content is directly responsible for altered microtubule dynamics in
tumor cells, resulting in altered sensitivity to taxanes.
Conversely overexpression of tubulin isotypes other than class I
may result from a compensation mechanism when class I genes are
mutated. In this respect it is noteworthy that in the mutations
reported by Monzo et al., a certain number corresponded to
premature termination mutations or frameshift mutations. However
the persistent expression of class I isotype in all tumor samples
analyzed in our series, including those with high class III levels,
does not support this hypothesis.
Microtubules are complex polymers which are susceptible to
post-translational alterations, including glutamylation and
phosphorylation of β tubulin, and acetylation or detyrosination of
alpha tubulin. The loss of the two terminal tyrosine residues by
alpha tubulin (“δ2 α tubulin”) has been shown to be correlated to
prolonged microtubule half-life in vitro or “stable” microtubules.
In our study only one tumor sample expressed δ2 α tubulin in tumor
cells. This is not in favor of enhanced microtubule stability as a
mechanism of resistance to taxanes. Other components shown to
participate in microtubular stabilization are MAPs, such as tau
protein. In preclinical models high level of tau protein were
correlated with in vivo resistance to docetaxel . Half of the
patients in our series expressed tau protein in their tumor cells,
but we did not find a correlation between the level of expression
of tau protein and response or patient survival.
Our results support the hypothesis that alteration in
microtubule components are responsible for resistance to taxanes in
lung cancer patients. Larger studies comparing sequencing data with
standardized immunohistochemical results and taking into account
other potentially important microtubule components are warranted.
These studies should further understanding into mechanisms of
resistance to taxanes and potentially provide opportunities to
improve the antitumor activity of taxanes in patients with
NSCLC.
Acknowledgements
Supported in part by the following grants (RFL): DAMD 17-98-1-8246
(US Army BCRP), CA26376 (National Institutes of Health), and
AQ-0726 (Welch Foundation).
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