Current use of cisplatin-based chemotherapy and surgery in the first-line treatment
of advanced testicular germ-cell tumours (TGCT) results in long-term disease-free
status in about 80% of cases (Bosl and Motzer, 1997; Horwich et al, 1998). As a result
of the high cure rate and the morbidity of treatment, clinical research has focused
on optimising the management of this disease, tailoring therapies according to the
risk of failure of the individual patient (Bosl and Motzer, 1997; Sonneveld et al,
2001). Factors associated with a poor-outcome have been analysed in several large
studies, and include the extent of metastatic disease and serum levels of the β-subunit
of human chorionic gonadotropin (β-hCG), α-fetoprotein (AFP) and lactate dehydrogenase
(LDH) (Germà-Lluch et al, 1980; Mead et al, 1992). The most recent and commonly employed
prognostic model for disseminated disease is the International Germ Cell Consensus
Classification (IGCCC), which predicts relapse at 5 years for 12, 25, and 59% patients
in the good, intermediate, and poor prognosis groups, respectively (International
Germ Cell Cancer Collaborative Group, 1997).
Treatment failure in this disease is closely related to inherent or developed resistance
to chemotherapy (Bosl and Motzer, 1997). Patients with refractory disease, failing
to respond to initial treatment, have a worse prognosis (Motzer et al, 1991). However,
current knowledge of the molecular determinants of chemoresistance involved in TGCT
is only marginal (Houldsworth et al, 1998; Rao et al, 1998; Kersemaekers et al, 2002).
In particular, little is known about clinical multidrug resistance (MDR) in TGCT (Van
Brussel and Mickisch, 1998). Multidrug resistance consists of de novo or acquired
cross-resistance to structurally and functionally unrelated drugs, some of them relevant
in the treatment of TGCT such as Vinca alkaloids, epipodophyllotoxins and anthracyclines
(Lehnert, 1996). Among the various molecular mechanisms associated with MDR in experimental
tumour models, one of the most extensively studied involves decreased drug accumulation
due to enhanced efflux by ATP-dependent transporter proteins such as P-glycoprotein
(Pgp), the human multidrug resistance-associated protein 1 (MRP1), and the recently
described breast cancer resistance protein (BCRP). P-glycoprotein overexpression is
associated with resistance to natural product drugs, including anthracyclines, etoposide,
vincristine and vinblastine, and paclitaxel (Germann, 1996). Multidrug resistance-associated
protein 1 is overexpressed in many non-Pgp-mediated MDR cell lines, conferring in
vitro resistance, among others, to etoposide, vincristine, vinblastine, and to methotrexate
after short-term exposure (Borst et al, 2000). Breast cancer resistance protein is
a newly described transporter, isolated from mitoxantrone-selected MDR cell lines
not expressing Pgp or MRP (Allikmets et al, 1998; Doyle et al, 1998). Breast cancer
resistance protein is associated with cross-resistance to dauno- and doxorubicin,
mitoxantrone, and camptothecins (Maliepaard et al, 1999).
Another protein related to an MDR-phenotype is the lung resistance-related protein
(LRP) (Scheper et al, 1993). Lung resistance-related protein has been identified as
the human major vault protein (MVP), the principal constituent of the complex ribonucleoprotein
particles known as vaults (Scheffer et al, 1995). Of interest in TGCT, LRP/vaults
have been associated with in vitro resistance to drugs such as etoposide, doxorubicin,
vincristine, and paclitaxel, but also to nonclassical MDR drugs such as cis-platin
and carboplatin (Izquierdo et al, 1996a; Kitazono et al, 1999). Although the function
of vaults in both normal and cancer cells is not fully elucidated, they are thought
to be involved in intracellular redistribution of drugs, reducing exposure of nuclear
targets from cytotoxic agents (Chugani et al, 1993; Kitazono et al, 1999).
We undertook the present study to assess the immunohistochemical expression of the
MDR-related proteins Pgp, MRP1, BCRP, and LRP in advanced TGCT, and to determine whether
such expression is related to response to first-line chemotherapy and survival.
PATIENTS AND METHODS
The expression of MDR-related proteins Pgp, MRP1, BCRP, and LRP was studied in paraffin-embedded
samples from patients with advanced TGCT. This expression was retrospectively correlated
with clinical and pathological characteristics of the patients, response to chemotherapy,
and outcome.
Sample selection
Between March 1990 and February 2001, 65 patients with primary advanced TGCT were
treated in our institution. We studied banked samples from 56 specimens, corresponding
to previously untreated patients undergoing orchidectomy for diagnostic evaluation
and treatment. Samples were obtained from the Department of Pathology at the Ciutat
Sanitària i Universitària de Bellvitge, Barcelona, Spain. Eligible patients were those
with advanced disease (stage I excluded) undergoing induction chemotherapy, and with
available material. The histological diagnosis was based on conventional morphologic
examination of paraffin sections, following World Health Organization criteria (Mostofi
and Sesterhenn, 1998).
Treatment, evaluation of response, and survival
All patients were primarily treated with inguinal orchidectomy and postoperative cisplatin-based
induction chemotherapy. Immediately after conventional induction chemotherapy, seven
poor-risk patients (IGCCC) received high-dose chemotherapy as consolidation treatment.
Complete responders to chemotherapy (CR) were patients with normalisation of serum
tumour markers and no clinical or radiological evidence of residual masses, or absence
of viable cancer cells such as seminoma, embryonal carcinoma (EC), immature teratoma,
yolk sac tumour (YS), choriocarcinoma (CC), and syncytiotrophoblastic cells in completely
resected residual masses (mature teratoma (MT) was not considered malignant component).
Complete responders to chemotherapy plus surgery (CR-S) were those with viable malignant
cells in completely resected residual masses. Incomplete responders (IR) were patients
with persisting elevation of tumour markers, incomplete surgical resection of residual
disease, or disease progression while receiving chemotherapy or within 1 month after
the completion of chemotherapy. Events in the progression-free survival (PFS) analysis
were: incomplete response, and relapse (rising tumour markers and/or an increase in
tumour volume, unless caused by completely resectable MT). Patients in progression
were managed with a variety of second-line chemotherapy regimens and surgery if appropriate.
Only deaths related to TGCT progression were considered as events for overall survival
(OS) analysis.
Monoclonal antibodies
The IgG1 murine monoclonal antibody (MAb) JSB-1 was used for Pgp detection in a concentration
of 50 μg ml−1 in phosphate-buffered saline (PBS) plus 1% bovine serum albumin (BSA,
Sigma, St Louis, MO, USA). For MRP1, the IgG2a MAb MRPm5 was used in a concentration
of 5 μg ml−1 in 1% PBS/BSA. For BCRP, the newly described IgG2a MAb BXP-21 was used
in a concentration of 25 μg ml−1 in 1% PBS/BSA. JSB-1, MRPm5, and BXP-21 were obtained
from the Department of Pathology, Free University Hospital, Amsterdam, the Netherlands
(Scheper et al, 1988; Flens et al, 1994; Maliepaard et al, 2001). None of these three
MAbs cross react with the other MDR proteins (Maliepaard et al, 2001). Finally, the
IgG1 murine MAb LRP (Transduction Labs, Los Angeles, CA, USA) was used for detection
of LRP in a concentration of 2.5 μg ml−1 in 1% PBS/BSA.
Immunohistochemistry
Immunohistochemistry was performed on 4 μm formalin-fixed, paraffin-embedded sections
mounted on poly-L-lysin-coated glass microslides, and dried at 37°C O/N. The immunoperoxidase
reaction conditions used for each MAb were selected on the basis of previous optimisation
of the immunohistochemical protocols. After conventional deparaffination and rehydration,
endogenous peroxidase activity was quenched by incubation in 3% H2O2 (10 min) at room
temperature. Pretreatment in a pressure cooker (20 min) with either citrate buffer
(10 mM citric acid pH 6.0 in distilled water) for MRPm5, LRP, and BXP-21, or EDTA
(1 mM) for JSB-1 was performed to unmask epitopes. Next, samples were incubated (30 min)
in normal rabbit serum (1 : 50 in 1% PBS/BSA), and then with the optimally diluted
specific antibody (60 min) at room temperature in a humidified chamber. The MAb BXP-21
was detected by the Dako EnVision™+System-horseradish peroxidase (Dako Corp., Glostrup,
Denmark) for 30 min (Diestra et al, 2002). A rabbit anti-mouse biotin-conjugated (1 : 150
for 30 min, Zymed Labs, San Francisco, CA, USA) followed by a streptavidin – horseradish
peroxidase (1 : 500 for 1 h, Zymed) method was used for MAbs MRPm5 and LRP. JSB-1
was developed by a twice-each alternating incubation with a rabbit anti-mouse biotin-conjugated
method (1 : 500 for 30 min, Dako) followed by a streptavidin–biotin complex (1 : 200
for 1 h, Dako). Staining with an irrelevant IgG or an isotype-specific antibody was
routinely performed as a negative control procedure in the normal tissues and in all
the clinical samples. Bound peroxidase was developed with 3,3′-diaminobenzidine (Sigma).
As positive control tissues for the immunohistochemical assays, liver was used for
JSB-1 (Van der Valk et al, 1990), colon for MRPm5 (Flens et al, 1996) and LRP (Izquierdo
et al, 1996b), and placenta for BXP-21 (Maliepaard et al, 2001). All slides were examined
and scored by two independent observers (EC and AZ) blinded to the clinical data.
Tumour cells were identified on morphological criteria. The MDR proteins were studied
in adjacent slides from the most representative paraffin block available for each
specimen. Only viable malignant germ cells (excluding MT elements) were considered
for evaluation. By prior agreement, in accordance with previous literature, sections
were evaluated in a semi quantitative way taking into account the percentage and intensity
of staining of tumour cells (Baldini et al, 1995; Izquierdo et al, 1995; Arts et al,
1999; Eid et al, 2000). Three staining categories were established: negative (no staining),
intermediate (positive staining in ⩽10% of tumour cells or weak and diffuse positive
staining), and strong (moderate/strong staining in >10% of tumour cells). A priori,
it was planned that the intermediate category needed to be grouped with either the
negative or the strong category for statistical correlations (see below). The reasons
for this were: (1) to ascertain whether the intermediate category provided more predictive
information for each protein when grouped with any of the other two categories; (2)
to perform meaningful statistical analysis with only two groups because of the limited
number of patients; (3) a clinical relevant cut-off level of expression of MDR proteins
is not well defined yet and it may be different for each protein and each tumour type.
Intensity and pattern of staining on the different histological subtypes in each sample
were recorded separately.
Statistical analysis
Clinicopathological characteristics were determined previous to chemotherapy. These
characteristics and response rates to induction chemotherapy were retrospectively
assessed for their relation with Pgp, MRP1, BCRP, and LRP expression, using χ
2 or the Fisher's exact two-tailed test as appropriate. For survival analysis, follow-up
started the date of the first cycle of induction chemotherapy. Univariate survival
analysis curves were generated using the Kaplan – Meier method and compared using
the log-rank test (Kaplan and Meier, 1958; Mantel, 1966). A stratified Mantel – Haenszel
test was performed to compare survival curves in order to adjust for the potential
confounding effect of statistically associated variables upon differences between
groups. Hypotheses were evaluated at a two-sided significance level of 0.05. The SPSS
package (SPSS Inc, Chicago, IL, USA) was used for calculations.
RESULTS
Clinical and pathological characteristics, response to chemotherapy, and outcome
The median age at initial orchidectomy was 24 years (range, 16–56 years). No patient
had a previous history of cancer, except for one (2%) having a contralateral and successfully
treated TGCT more than 9 years before. The characteristics of the 56 patients included
in the study are summarised in Table 1
Table 1
Clinical and pathological characteristics according to Pgp, MRP1, BCRP, and LRP expression
No. (%)
Pgp+(%)a
MRP1+(%)a
BCRP+(%)a
LRP+(%)a
All cases
56(1 0 0)
15(27)
30(54)
48(86)
24(43)
Histology
Nonseminoma
52(93)
13(25)
27(52)
46(88.5)
23(44)
Seminoma
4(7)
2(50)
3(75)
2(50)
1(25)
Stage (Royal Marsden)
IM+II–III
39(70)
12(31)
20(51)
32(82)
13(33)
IV
17(30)
3(18)
10(59)
16(94)
11(65)***
Pre-CT serum TM
AFP
⩽1000 ng ml−1
45(80)
15(33)*
23(51)
39(87)
19(42)
>1000 ng ml−1
11(20)
0(0)
7(64)
9(82)
5(45.5)
β-hCG
⩽5000 IU l−1
47(84)
13(28)
24(51)
40(85)
16(34)
>5000 IU l−1
9(16)
2(22)
6(67)
8(89)
8(89)****
LDH
⩽1.5 upper N thresh
46(82)
13(28)
21(46)
39(85)
19(41)
>1.5 upper N thresh
10(18)
2(20)
9(90)**
9(90)
5(50)
IGCCC
Good
35(62.5)
11(31)
16(46)
30(86)
13(37)
Intermediate or poor
21(37.5)
4(19)
14(67)
18(86)
11(52)
Vascular invasion
Yes
38(68)
12(32)
22(58)
34(89)
17(45)
No
18(32)
3(17)
8(44)
14(78)
7(39)
CT=chemotherapy, N thresh=normal threshold, TM=tumour markers.
a
Pgp+, MRP1+, and BCRP+ represent immunohistochemical category strong; LRP+ represents
the grouping of categories intermediate and strong (see Patients and Methods, and
Results).
*
P=0.026 (Fisher's exact test).
**
P=0.014 (Fisher's exact test).
***
P=0.029 (χ2 test).
****
P=0.003 (Fisher's exact test).
(left column). The 52 non-seminomatous TGCT included: 41 mixed tumours, seven EC,
three YS, and one CC. Thirty-five (62%) were good-risk patients, 11 (20%) intermediate-risk,
and 10 (18%) poor-risk, according to the IGCCC criteria (International Germ Cell Cancer
Collaborative Group, 1997). Chemotherapy regimens used (Germà-Lluch et al, 1999) are
listed in Table 2
Table 2
First-line chemotherapy regimens
Patients (N=56)
Regimens
No.
%
BEPa
37
66
BOMP-EPI
16
29
EP
2
4
Other platinum based
1
1
High doseb
7
13
BEP=etoposide, cisplatin, bleomycin, EP=etoposide, cisplatin, BOMP-EPI=bleomycin,
vincristine, methotrexate, cisplatin/etoposide, ifosfamide, cisplatin.
a
Includes one patient nonassessable for response.
b
Immediately after conventional induction chemotherapy, seven poor-risk patients (IGCCC)
received high-dose chemotherapy as consolidation treatment.
. Median number of cycles was 4 (range, 2–8). One patient (2%) was excluded from the
analysis of response to chemotherapy because of a nonrelated-to-cancer death during
treatment. Therefore, 55 patients were evaluable for tumour response to induction
chemotherapy. Fifty-one CR (93%), three CR-S (5%), and one IR (2%) to first-line chemotherapy
were accounted. Among those achieving a CR, 16 underwent surgery of residual masses:
six showed either necrosis or fibrosis and 10 MT only. One of the CR-S patients actually
progressed to first-line chemotherapy but underwent complete salvage surgery and continues
relapse-free with more than 4 years of follow-up.
The median follow-up was 49.5 months (range 2–111 months). Seven patients (12.5%)
progressed to first-line treatment, including 1 IR and 6 relapses. Four patients (7%)
died of TGCT and two (4%) of non-TGCT-related reasons (second carcinoma and intracranial
haemorrhage).
Pgp, MRP1, BCRP, and LRP expression in TGCT at diagnosis
The results of immunohistochemistry are summarised in Table 3
Table 3
Pgp, MRP1, BCRP, and LRP expressions in testicular germ-cell tumours at diagnosis
Category
Stronga
Intermediatea
Negativea
Pgp
15 (27%)
23 (41%)
18 (32%)
MRP1
30 (54%)
21 (37%)
5 (9%)
BCRP
48 (86%)
4 (7%)
4 (7%)
LRP
8 (14%)
16 (29%)
32 (57%)
a
Strong: strong staining in >10% of tumour cells; intermediate: positive staining in
⩽10% of tumour cells or weak and diffuse positive staining; negative: no staining.
. Fifteen (27%) and 23 (41%) of the 56 specimens showed strong and intermediate expression
of Pgp, respectively. The staining pattern was diffuse and homogeneous, cytoplasmic
and occasionally membranous. Thirty (54%) and 21 (37%) of the 56 cases displayed strong
and intermediate expression of MRP1, respectively. Immunostaining was rather heterogeneous.
MRP1 was detected at the cytoplasm, although nuclear and scarce membranous expressions
were also found. For both Pgp and MRP1, the intermediate category was composed mainly
of samples with a weak expression in over 10% of tumour cells (20 out of 23 and 17
out of 21, respectively). Forty-eight (86%) and four (7%) of the 56 cases displayed,
respectively, strong and intermediate expression of BCRP, with a mixed staining pattern,
membranous (particularly among EC and YS elements) and cytoplasmic (in syncytiotrophoblasts).
BCRP staining intensity was mostly strong and rather homogeneous as shown in Figure
1A
Figure 1
Immunohistochemical staining on paraffin-embedded specimens of testicular germ-cell
tumours. (A) (× 100) BCRP strong positive embryonal carcinoma stained with MAb BXP-21.
Note the strong diffuse membranous and cytoplasmic staining pattern. (B) (× 200) LRP
strong positive choriocarcinoma stained with MAb LRP. Note the strong granular cytoplasmic
pattern in syncytiotrophoblastic cell.
, except for YS elements (usually positive in areas with epithelial differentiation
and negative where mesenchymal). Eight (14%) and 16 (29%) of the 56 cases displayed,
respectively, strong and intermediate expression of LRP, with a typical granular cytoplasmic
pattern as shown in Figure 1B (Izquierdo et al, 1996b). Intensity was mostly strong
and heterogeneous, with the exception of syncytiotrophoblastic areas, which showed
homogeneous staining. Remarkably, 15 of 16 samples in the intermediate category had
less than 10% intense positive cells. In eight of these 15, one histological subtype
(six CC and two immature teratomas) with a minor overall representation (⩽10%) was
found universally positive. No staining was observed among negative controls for all
four MAbs. No correlation was found between the expressions of the different proteins
(data not shown).
MDR status and established prognostic factors
For the reasons described before (see Patients and Methods), the intermediate immunohistochemical
category was grouped either with the negative or the strong category for statistical
correlations with other prognostic factors and analysis of outcome. Table 1 summarises
these correlations. Tumours with strong expression of Pgp were more frequently observed
in patients with AFP levels <1000 ng ml−1 (P=0.026). Tumours with strong expression
of MRP1 correlated with LDH levels >1.5 upper normal threshold (P=0.014). No association
could be identified for BCRP. Tumours with strong or intermediate expression of LRP
associated with β-hCG levels >5000 IU/l−1 (P=0.003) and stage IV (P=0.029) according
to the Royal Marsden Hospital (RMH) Classification (Peckham et al, 1979).
MDR status and response to induction chemotherapy
Regardless of the grouping of the intermediate category, no significant association
was observed between the expression of Pgp, MRP1, BCRP, and LRP and the response to
induction chemotherapy (Table 4
Table 4
Expression of Pgp, MRP1, BCRP, and LRP and response to induction chemotherapy (n=55
evaluable patients)
n
CR (%)
CR-S + IR (%)
P
a
Pgp
b
+
14
12(86)
2(14)
0.265
−
41
39(95)
2(5)
MRP1
b
+
29
26(90)
3(10)
0.613
−
26
25(96)
1(4)
BCRP
b
+
47
44(94)
3(6)
0.477
−
8
7(87.5)
1(12.5)
LRP
b
+
23
21(91)
2(9)
0.999
−
32
30(94)
2(6)
CR=complete response to chemotherapy, CR-S=complete response to chemotherapy and surgery,
IR=incomplete response (see Patients and Methods).
a
Fisher's exact test.
b
Pgp+, MRP1+, and BCRP+ represent immunohistochemical category strong; Pgp-, MRP1-,
and BCRP- represent the grouping of intermediate and negative categories. The grouping
of strong and intermediate categories resulted also in not significant correlations
(data not shown) (see Patients and Methods, and Results). LRP+ represents the grouping
of categories intermediate and strong; LRP- represents negative category. The grouping
of intermediate and negative categories resulted also in not significant correlations
(data not shown). For LRP grouping of categories intermediate and strong is shown
due to the prognostic value on PFS and OS (see Patients and Methods, and Results).
).
MDR status and clinical outcome
Three-year PFS for all 56 patients included in the survival analysis was 85.3% (95%
CI, 75.2–95.3%), and 3-year OS 91.4% (83.9–99.4%). With seven relapses and four cancer-related
deaths, median PFS and OS have not been reached. Regardless of the grouping of the
intermediate category, no association was observed for Pgp, MRP1, and BCRP expressions
either with PFS (strong vs intermediate/negative: P=0.307, 0.284, and 0.284, respectively)
or OS (strong vs intermediate/negative: P=0.932, 0.98, and 0.053, respectively). For
all three MDR proteins, the grouping of the strong plus intermediate category resulted
in more unbalanced arms (very few patients in the negative category) and in lack of
significant correlations (data not shown). In contrast, patients whose tumours showed
intermediate or strong expressions of LRP had significantly shorter PFS (P=0.0006;
Figure 2A
Figure 2
Progression-free survival according to LRP expression previous to treatment in 56
patients with advanced testicular germ-cell tumours. (A) Immunohistochemical categories
strong and intermediate (grouped) vs negative. (B) Considering the three original
immunostaining categories used for scoring (see Patients and Methods, and Results).
) and OS (P = 0.0116; Figure 3
Figure 3
Overall survival according to LRP expression previous to treatment in 56 patients
with advanced testicular germ-cell tumours. Immunohistochemical categories: strong
and intermediate (grouped) vs negative.
) than LRP-negative patients. When considering the three original immunostaining categories
separately, log-rank statistics for PFS and OS were P=0.0008 and 0.035, respectively
(Figure 2B). The grouping of the intermediate and strong categories discriminated
better the outcome according to LRP expression. By univariate analysis, LRP was the
strongest adverse prognostic marker for PFS (P=0.0006) and OS (P=0.0116), followed
by β-hCG level >5000 IU l−1 previous to induction chemotherapy (P=0.039 for PFS, and
P=0.059 for OS). Risk stratification according to the IGCCC (intermediate-poor vs
good) or RMH (stage IV vs IM–II – III) did not achieve prognostic significance either
for PFS (P=0.07 and 0.481, respectively) or OS (P=0.138 and 0.38, respectively) in
this relatively small set of patients. As no recurrences or cancer-related deaths
(i.e. events) were accounted among LRP-negative patients, no multivariate Cox regression
analysis could be performed. Instead, we tested survival distributions for LRP adjusting
for statistically related variables performing a stratified Mantel–Haenszel test.
LRP remained significant after adjusting for β-hCG level (P=0.0029 for PFS, and P=0.038
for OS), and RMH stage (P=0.001 for PFS, and P=0.02 for OS).
DISCUSSION
The increasing need of new markers for prediction of response to chemotherapy and
long-term PFS (the most relevant end point for TGCT patients in prognostic factor
analyses), and the scarce data concerning the expression and significance of MDR-related
proteins in TGCT, prompted us to initiate this study.
The most remarkable finding in our series of advanced TGCT was that LRP immunoreactivity
in pretreatment tumour cells was prognostic of the clinical outcome. LRP immunostaining,
at any level, was detected in 43% of the patients, consistent with the 50% previously
reported in a short series with only 12 specimens (Izquierdo et al, 1996b). LRP is
the human major protein of cellular organelles known as vaults (Scheffer et al, 1995;
for review, see Scheffer et al, 2000b), ribonucleoprotein particles with a peculiar
ovoid structure that is highly conserved among various species. Vaults have been localised
in the cytoplasm but also in the nuclear membrane, probably at the nuclear pore complex
(Chugani et al, 1993). They have been found broadly distributed in normal human tissues
and in tumours (Izquierdo et al, 1996b). Although vault function is yet undetermined,
they are thought to mediate vesicular and nucleo-cytoplasmic trafficking and transport
processes of various substrates including cytostatics (Chugani et al, 1993; Kitazono
et al, 1999). The relevance of LRP as a constituent of the whole vault particle in
the prediction of a MDR phenotype is well documented in vitro in numerous human cancer
cell lines (Scheper et al, 1993; Izquierdo et al, 1996a; Laurençot et al, 1997; Kickhoefer
et al, 1998). Kitazono et al (1999) recently provided the first evidence favouring
a causal relation between vaults and drug resistance. Moreover, basal LRP expression
has been found indicative for cisplatin and carboplatin resistance (nonclassical MDR-related
drugs) in the NCI panel of 61 unselected human tumour cell lines used for drug screening
(Izquierdo et al, 1996a), and in nonsmall-cell lung cancer cell lines (Berger et al,
2000).
In our study, any extent of LRP immunoreactivity including strong expression in a
minority of cancer cells was associated with an unfavourable prognosis even after
individual log-rank adjustments for statistically related prognostic factors. The
strong biological and clinical rationale supporting the association of LRP with resistance
to a broad spectrum of anticancer drugs, including cisplatin, and the fact that the
two correlations found go in the same direction (LRP expression correlates with both
shorter PFS and OS), makes unlikely that the positive analyses are due to multiple
subset analysis. The frequent presentation of TGCT as mixed histologies and the potential
significance for prognosis of a minor component in them make this a unique entity.
Our finding adds to several clinical studies reporting overexpression of LRP at diagnosis
as an independent predictor for clinical outcome and/or chemotherapy failure, for
example, in acute myeloid leukaemia (List et al, 1996; Borg et al, 1998; Filipits
et al, 1998,2000), multiple myeloma (Raaijmakers et al, 1998; Filipits et al, 1999),
stage III–IV ovarian carcinoma (Izquierdo et al, 1995), and locally advanced bladder
cancer (Diestra et al, 2001). Other studies have failed to show prognostic values
of LRP immunoreactivity in acute leukaemia (Damiani et al, 1998; Leith et al, 1999)
and ovarian carcinoma (Arts et al, 1999). Despite the prognostic value of LRP in this
study, no relation was observed between LRP expression and response to platinum-based
chemotherapy. Whether this lack of correlation was because of the very high rate of
CR observed (93%, otherwise expected in TGCT) in our relatively small series of patients
remains uncertain. As an alternative explanation, LRP may be a marker of biological
aggressiveness in TGCT. This is suggested not only by the fact of poorer clinical
outcomes for patients with LRP-expressing tumours, but also by the observed correlation
between LRP and well-known factors conferring bad prognosis such as high β-hCG levels
and visceral metastatic disease. Baldini et al (1995) found a similar conclusion for
Pgp in high-grade osteosarcomas. They reported a strong correlation between the presence
of increased levels of Pgp at diagnosis and bad prognosis that was unrelated to response
to chemotherapy. We cannot exclude a potential influence of the histological subtype
CC over the observed LRP value for prognosis in our series, as all of the CC elements
stained for LRP (and β-hCG is elaborated by syncytiotrophoblastic components in CC).
However, most prognostic classifications have failed to identify any particular histology
as an independent variable for outcome (Mead et al, 1992). More detailed studies on
the relation of LRP with clinicopathological parameters in TGCT are therefore warranted.
Pgp expression was observed in 27% of tumours, in agreement with previous reports
(35%, Katagiri et al, 1993; 33%, Eid et al, 1998). In contrast to the report by Eid
et al (1998), we found no association between Pgp expression and nonseminomatous histology
or advanced stages of disease. This discrepancy can be attributed to our small number
of seminomas and the use of different methodologies for Pgp detection. In agreement
with the previous study, Pgp expression did not predict response to chemotherapy and
was not related to survival. Neither in vitro nor clinical data suggest a relation
between Pgp expression and resistance to cisplatin. However, the incorporation of
etoposide, a substrate for Pgp (Germann, 1996), into combination chemotherapy regimens
has improved response rates and long-term survival of poor-risk TGCT patients (Bosl
and Motzer, 1997; Sonneveld et al, 2001). Whether the expression of Pgp in TGCT influences
response to chemotherapy and outcome after regimens containing etoposide needs to
be investigated in a much larger group of patients primarily treated with this drug.
There is only one previous study concerning MRP1 expression in TGCT (Eid et al, 2000).
We report MRP1 overexpression in 54% TGCT, whereas Eid et al (2000) reported in 100%
using a different MAb but similar criteria for assessment. In our study, MRP1 expression
did not predict response to chemotherapy or outcome. All attempts to demonstrate MRP1
as a mediator of cisplatin resistance have failed thus far; although MRP1 is involved
in efflux conjugates of drugs to glutathione (one of the cellular thiol compounds
involved in resistance to platinum agents; Borst et al, 2000). Eid et al (2000) reported
MRP1 expression in the nucleus, in accordance with our observation, an intriguing
binding of unclear significance. They speculated about a possible role for MRP1 unique
to TGCT in the intracellular redistribution of drugs. However, we cannot exclude this
finding being a staining artefact.
Recently, the novel ATP-dependent transporter BCRP was identified in mitoxantrone-selected
MDR cell lines not expressing Pgp or MRP1 (Allikmets et al, 1998; Doyle et al, 1998).
Thus far, BCRP has been described in cancer cell lines of different histogenetic origin
(Scheffer et al, 2000a). Additionally, expression of BCRP has been reported in normal
human tissues (Maliepaard et al, 2001), in a small panel of human tumour samples including
two seminoma specimens (both considered BCRP-negative; Scheffer et al, 2000a), and
in human breast carcinoma (Kanzaki et al, 2001) and acute leukaemia (Ross et al, 2000).
We report BCRP overexpression in 86% of TGCT, particularly in EC and in syncytiotrophoblasts
in CC. The high expression rate of BCRP in placental syncytiotrophoblast (Maliepaard
et al, 2001) is therefore retained in its malignant counterpart. BCRP did not predict
response to chemotherapy and was not related to survival, consistent with its wide
expression in TGCT and the limited role in this disease of the cytostatics to which
BCRP confers resistance. In particular, BCRP is not involved in resistance to cisplatin
(Allikmets et al, 1998; Doyle et al, 1998; Maliepaard et al, 1999).
In conclusion, our data suggest that the expression of LRP at the time of diagnosis
of a metastatic TGCT may identify a patient population with a tendency to progress
despite chemotherapy. The adverse prognostic value of LRP may be important because
it may influence early selection of TGCT patients for novel therapeutic strategies.
Moreover, the search for pharmacological agents capable of affecting vault function
might also help in optimising treatment protocols in the future. However, because
of the limited number of patients in this study, our results must be interpreted with
caution. Whether LRP will increase the predictive power of current clinically oriented
prognostic models remains to be determined in larger, prospective studies. The intriguing
hypothesis derived from our study may prompt the initiation of such studies by institutions
or collaborative groups capable of recruiting a large number of cases of this relatively
rare type of cancer. We strongly encourage confirmation or rebuttal of our findings
(especially the zero cell) by others.