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976
Multidrug Resistance Phenotype in High Grade Soft
Tissue Sarcoma
Correlation of P-Glycoprotein Immunohistochemistry with Pathologic Response
to Chemotherapy
Rafael E. Jimenez, M.D.1
Mark M. Zalupski, M.D.2
John J. Frank, M.T.1
Wei Du, Ph.D.2
James R. Ryan, M.D.3
David R. Lucas, M.D.1
1
Department of Pathology, Harper Hospital,
Wayne State University and the Karmanos Cancer
Institute, Detroit, Michigan.
2
Department of Oncology, Harper Hospital, Wayne
State University and the Karmanos Cancer Institute, Detroit, Michigan.
3
Department of Orthopedics, Hutzel Hospital,
Wayne State University and the Karmanos Cancer
Institute, Detroit, Michigan.
BACKGROUND. P-glycoprotein–mediated drug efflux has been implicated as an
important mechanism of multidrug resistance (MDR) in cancer. Its role in chemotherapy resistance in soft tissue sarcoma is unclear.
METHODS. Tumor specimens prior to and following neoadjuvant chemotherapy
from 29 cases of high grade soft tissue sarcoma were analyzed with 2 monoclonal
antibodies (C494 and JSB-1) that recognize different epitopes of P-glycoprotein.
Staining intensity was graded 0 5 negative, 1 5 equivocal, 2 5 moderate, 3 5
strong. Only cases with Grade 2 or 3 staining intensity with both antibodies were
considered MDR positive. The resection specimens were evaluated for tumor
necrosis postchemotherapy. Pathologic response was graded as good for ,15%,
moderate for 15–50%, or poor for .50% posttreatment tumor viability.
RESULTS. Of the 29 pretreatment specimens, 10 (34%) were MDR positive and 19
(66%) were MDR negative. Pathologic response to treatment was characterized as
good in 6, moderate in 7, and poor in 16 patients. Of the MDR positive biopsies, 9
(90%) had poor response, compared with 7 (36%) in the MDR negative biopsy
group (P 5 0.0078). None of the cases with MDR positive biopsies had a good
response, compared with 6 cases in which biopsies were MDR negative (32%) (P 5
0.057). Only one MDR negative case became MDR positive posttreatment.
CONCLUSIONS. Expression of MDR phenotype is found in approximately one-third
of high grade soft tissue sarcomas. These preliminary data show a significant
correlation between MDR phenotype and poor pathologic response to chemotherapy, and suggest that MDR induction by chemotherapy in soft tissue sarcoma is an
uncommon event. Cancer 1999;86:976 – 81. © 1999 American Cancer Society.
KEYWORDS: soft tissue sarcoma, chemotherapy, chemoresistance, MDR, P-glycoprotein, JSB-1, C494.
P
Presented at the 87th Annual Meeting of the United
States and Canadian Academy of Pathology, Boston, Massachusetts, February 28 to March 6,
1998.
Address for reprints: David R. Lucas, M.D., Department of Pathology, Harper Hospital, 3990 John R,
Detroit, MI 48201.
Received October 15, 1998; revision received February 10, 1999; accepted April 7, 1999.
© 1999 American Cancer Society
-glycoprotein (Pgp) expression has been implicated as a main
cause for multidrug resistance (MDR) in several human neoplasms.1 This phenomenon is defined as an intrinsic or acquired
resistance to multiple anticancer drugs that appear to be structurally
and functionally unrelated. Pgp, the product of the mdr1 gene, localized in the long arm of chromosome 7, is a 170 kD cell membrane
protein that actively pumps out drugs through a transmembrane
channel. It is expressed in normal tissues, particularly in specialized
epithelial cells with secretory functions, such as hepatocytes, colonic
mucosa, biliary and pancreatic ductules, and brush border of the
proximal tubules of the kidney, among others. The main characteristic of this protein is its broad substrate specificity, which encompasses a large list of anticancer drugs, including doxorubicin, an agent
MDR in Soft Tissue Sarcoma/Jimenez et al.
widely used in sarcoma treatment.2,3 Overexpression
of Pgp has been shown to correlate with chemoresistance in a variety of cancers.4 –13
In addition, induction of Pgp expression by chemotherapeutic agents has also been reported,7,14,15
suggesting an explanation for failure of treatment in
tumors initially MDR negative.
Although its effectiveness is still somewhat controversial, neoadjuvant chemotherapy is widely used
in the treatment of adult soft tissue sarcoma (STS).16,17
Multiple clinical trials have suggested a better outcome for treated patients,2,3 although the success obtained with osteosarcoma18,19 or pediatric soft tissue
tumors13,14 has not been achieved.
To what extent MDR expression determines the
response to chemotherapy in various soft tissue neoplasms has been a subject of investigation in multiple
recent studies.13,14,20 –26 The role that it plays specifically in adults STS is still not completely understood,
and contradictory data have emerged recently.
In this study we investigated expression of MDR
by immunohistochemical methods, utilizing two
monoclonal antibodies that identify different epitopes
of Pgp. We studied pretreatment and posttreatment
specimens from 29 cases of a variety of high grade STS,
and the data were correlated with pathologic response
to chemotherapy. We found expression of MDR phenotype in approximately one-third of cases, as well as
significant correlation between MDR phenotype and
poor pathologic response to therapy.
977
case containing diagnostic tissue was chosen to perform additional stains.
Immunohistochemistry
Slides for immunohistochemical studies were stained
with C494 (Signet Laboratories, Dedham, MA; 1:100
dilution of a 0.100 mg/mL concentrate mouse monoclonal, 20-minute incubation) and with JSB-1 (Signet
Laboratories; 1:10 dilution of a 0.20 mg/mL concentrate mouse monoclonal, overnight incubation). Vimentin staining was performed on the Ventana Medical Systems ES automated slide stainer using
prediluted antibody (clone 3B4, Ventana Medical Systems, Tucson, AZ). Sections of kidney, liver, adrenal
gland, and colonic mucosa were used as positive controls for C494 and JSB-1. Negative controls were run
on every sample.
Scoring of Immunostaining
Tissue immunoreactivity was evaluated with vimentin
immunostaining. Staining intensity for C494 and JSB-1
was scored as 0 (negative), 1 (equivocal), 2 (moderate),
or 3 (strong). Cytoplasmic (Fig. 1), membranous (Fig.
2), or Golgi region (Fig. 3) staining were considered
positive. To avoid false-positive results due to ambiguous and/or excessive background staining, only
moderate or strong staining (Grades 2 and 3) were
considered positive. Only cases positive for both antibodies were considered MDR positive.
Statistical Analysis
METHODS
General
Paraffin embedded tissue blocks from 29 patients with
adult STS, including pretreatment and posttreatment
specimens (58 cases), were retrieved. Clinical and
pathologic information was obtained from medical
records and surgical pathology reports.
Pathology
Hematoxylin and eosin (H&E) diagnostic materials
were reviewed by two pathologists (R.E.J., D.R.L.) with
a double-headed microscope. The cases were evaluated to corroborate histologic type and grade of sarcoma and to assess pathologic response to chemotherapy. Histologic grade was determined in the
pretreatment specimen according to cellularity, mitotic activity, nuclear hyperchromasia, pleomorphism,
and necrosis. A four-tier grading system was used.
Pathologic response was evaluated according to the
degree of necrosis present in the posttreatment resection specimens, and was considered good for ,15%,
moderate for 15–50%, and poor for .50% of tumor
viability. At least one representative block from each
Comparisons between groups were made with the
Fisher exact test.
RESULTS
There were 12 women and 17 men in the study population. The mean age was 52.2 6 16.2 years; the
median age was 56 years. All patients were treated
between 1990 and 1998 and received 3 cycles of neoadjuvant chemotherapy consisting of doxorubicin,
dacarbazine, and ifosfamide, delivered by infusion
over 72–96 hours. Cycles were generally repeated at
21-day intervals, with the last treatment completed
3–5 weeks prior to surgical resection. The histologic
type of sarcoma included 12 malignant fibrous histiocytomas, 5 synovial sarcomas, 3 liposarcomas, 3 malignant peripheral nerve sheath tumors, 1 rhabdomyosarcoma, 2 leiomyosarcomas, 1 fibrosarcoma, 1
myxoid chondrosarcoma, and 1 undifferentiated sarcoma. All cases had a histologic Grade 3 (n 5 8) or 4
(n 5 21) out of 4. Twenty-two tumors were localized in
the extremities and 7 in the trunk. The mean size of
the tumors was 11.9 6 7.4 cm.
Among the 29 pretreatment specimens, 10 (34%)
978
CANCER September 15, 1999 / Volume 86 / Number 6
FIGURE 1. (A) Intracytoplasmic immunopositivity with JSB-1 antibody is shown in a malignant peripheral nerve sheath tumor (original magnification 3100). (B)
Diffuse positivity with C494 antibody is noted in this monophasic synovial sarcoma. The pattern of staining is cytoplasmic (original magnification 350).
FIGURE 2. (A) This myxoid chondrosarcoma (chordoid sarcoma) displays prominent membranous staining with C494 antibody (original magnification 3100). (B)
Prominent membranous and cytoplasmic pattern of immunostaining is shown in a rhabdomyosarcoma with C494 antibody (original magnification 3100).
were positive for both C494 and JSB-1, and thus were
considered MDR positive (95% confidence interval,
0.184 – 0.549). Twelve cases were negative for both
markers, and 7 cases were positive with only 1 marker
(Table 1). Thus, a total of 19 cases (66%) were considered MDR negative (95% confidence interval, 0.451–
0.816).
Sixteen patients had a poor pathologic response, 7
had a moderate response, and 6 had a good response
to preoperative chemotherapy. Of the cases with MDR
positive biopsies, 9 (90%) had a poor response (95%
confidence interval, 0.603– 0.995) and 1 (10%) had a
moderate response (Table 2). In the MDR negative
biopsy group, 7 (36%) had a poor response (95% confidence interval, 0.150 – 0.635) (P 5 0.0078) and 13
(68%) had a poor or moderate response (P 5 0.057)
(i.e., none of MDR positive cases had a good response,
compared with 32% MDR negative cases). No signifi-
FIGURE 3. Golgi staining pattern with JSB-1 is seen in this malignant
peripheral nerve sheath tumor (original magnification 3250).
MDR in Soft Tissue Sarcoma/Jimenez et al.
979
TABLE 1
Summary of Clinical Data and Pathologic Findings
Case
Age (yrs)
Gender
Diagnosis
Location
JSB-1a
C494a
Response
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
74
65
40
55
56
56
47
23
39
36
70
68
52
29
40
42
70
65
75
21
62
56
18
62
39
64
70
61
68
M
M
M
M
M
M
M
F
M
F
M
F
F
F
M
M
F
F
M
F
F
M
M
F
M
F
M
F
M
Leiomyosarcoma
MFH
MPNST
MPNST
Myxoid chondrosarcoma
Myxoid liposarcoma
Synovial sarcoma
Synovial sarcoma
Synovial sarcoma
MFH
MFH
MFH
MPNST
Synovial sarcoma
Synovial sarcoma
MFH
MFH
MFH
MFH
Undifferentiated sarcoma
Pleomorphic liposarcoma
Fibrosarcoma
Alveolar rhabdomyosarcoma
Epithelioid leiomyosarcoma
Round cell liposarcoma
MFH
MFH
MFH
MFH
R thigh
L thigh
R arm
L axilla
Paraspinal
R thigh
L thigh
L thigh
L thigh
L thigh
R thigh
L thigh
R forearm
R shoulder
R groin
R forearm
L buttock
R thigh
L forearm
L thigh
L thigh
L buttock
L thigh
L thigh
R thigh
R thigh
R groin
L thigh
L forearm
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
1
2
2
2
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
1
1
2
2
2
1
1
1
1
1
2
2
2
2
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Poor
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Good
Good
Good
Good
Good
Good
MFH: malignant fibrous histiocytoma; MPNST: malignant peripheral nerve sheath tumor.
a
Positivity in pretreatment specimen.
TABLE 2
MDR Positivity vs. Pathologic Response
MDR positive
MDR negative
Total
Good
Moderate
Poor
Total
0
6 (32%)
6 (21%)
1 (10%)
6 (32%)
7 (24%)
9 (90%)
7 (36%)
16 (55%)
10
19
29
MDR: multidrug resistance.
cant correlation was observed between histologic
type, grade, or any histologic feature and response.
Only in one case with an MDR negative pretreatment specimen was Pgp expressed in the resection
specimen, suggesting a low incidence of MDR phenotype induction by chemotherapy.
DISCUSSION
MDR, as defined by Pgp overexpression, has been
extensively implicated in chemotherapy resistance in
numerous human cancers, particularly in neoplasms
derived from tissues that normally express Pgp (i.e.,
colonic and renal carcinomas).1 However, the list of
neoplasms in which MDR is believed to play a significant role in chemoresistance is increasing. In addition, MDR phenotype induction has been documented in neoplasms that did not express it prior to
chemotherapy.7,14,15
Recently, the question of whether chemoresistance in adult STS could be explained by Pgp expression has been raised.14,20 –26 Recent data have suggested a strong correlation between Pgp expression
and outcome following therapy in pediatric STS13,14
and osteosarcoma.19,27 However, few studies have
looked into prediction of response of adult STS to
chemotherapy.
The reported incidence of Pgp overexpression in
STS ranges from 0% to 100%.20 This large range of data
is probably best explained by the multiple techniques
utilized in the evaluation of either mdr1 expression
(mRNA) or Pgp detection, including slot or dot blot,
980
CANCER September 15, 1999 / Volume 86 / Number 6
Northern blot, reverse transcriptase–polymerase chain
reaction, RNase protection assay, in situ hybridization, immunoblot, and immunohistochemistry. With
so many techniques and criteria used to assess MDR
phenotype, results are difficult to compare.
Immunohistochemical studies such as ours also report variable incidences, which is probably a reflection
of the different numbers and clones of monoclonal antibodies and the variety of criteria utilized for interpretation. For example, Nakanishi et al.22 used staining with
either of two antibodies as criterion for MDR positivity
and reported an incidence of 62% of MDR phenotype
expression in a variety of STS. On the other hand, Vergier
et al.23 considered only 1 of 22 cases of STS to be MDR
positive, using immunostaining with all of 3 different
antibodies as the criterion for MDR positivity.
Advantages of immunohistochemical assessment
of Pgp expression over other techniques include accessibility to archival fixed tissue, the ability to distinguish between tumor cell expression and normal cell
expression, assessment of Pgp directly, and relatively
good sensitivity. Limitations of the technique, however, include control of fixation of the tissue and
cross-reactivity of monoclonal antibodies with other
non-Pgp-related proteins.14,23,28 In our study, adequate immunoreactivity was assessed by staining of
the tissue with vimentin, indirectly reflecting an acceptable degree of fixation of the tissue. To address
the issue of cross-reactivity, several authors, including
a consensus panel recommendation,28 suggest that at
least two different monoclonal antibodies directed toward two different epitopes of Pgp should be used to
determine MDR positivity by immunohistochemistry.
Our criterion for MDR positivity was unequivocal
staining of tumor cells with both monoclonal antibodies utilized, thus diminishing the likelihood of falsepositive staining.
The clinical significance of MDR positivity in
STS is uncertain. Data from published studies are
difficult to compare, due to the high variability of
clinical outcome parameters (clinical response,
pathologic response, and relapse free and overall
survival) and modalities of treatment employed
(mono/polychemotherapy, surgery, and/or radiation).20 Chan et al.13 found that positive staining for
Pgp was a strong predictor of adverse outcome and
poor response to chemotherapy in pediatric STS.
Nakanishi et al.22 found a significant difference in
the 5-year survival of patients with Pgp positive
tumors (51%) versus Pgp negative tumors (84%).
Conversely, Leuschner et al.21 and Stein et al.24
could not demonstrate any correlation between immunohistochemistry and treatment response.
Our data show a significant correlation between
MDR positivity, as defined by immunohistochemical
detection of Pgp, and pathologic response to chemotherapy. Ninety percent of the patients with MDR
positive tumors had a poor pathologic response, compared with only 36% of MDR negative cases. Although
based on a relatively small number of cases with heterogeneous histologic types, these results suggest a
role for MDR in resistance to chemotherapy in high
grade STS. However, 42% of cases with poor response
(7 of 16) were MDR negative, which suggests that other
factors besides MDR phenotype may contribute to STS
chemoresistance.
Induction of MDR in neoplastic cells by chemotherapeutic agents has been a matter of concern expressed
in several recent publications.7,14,15 Our data show that
only one pretreatment MDR negative case expressed
MDR phenotype after chemotherapy in the resection
specimen. This information suggests that MDR phenotype induction by chemotherapeutic agents in the setting of adult STS is an infrequent event.
CONCLUSIONS
MDR phenotype, as determined by immunohistochemical detection of Pgp, is expressed in approximately one-third of high grade adult STS. These preliminary data show a significant correlation between
MDR phenotype and poor pathologic response to chemotherapy. Further studies are needed to confirm the
role of MDR phenotype and other possible mechanisms in the chemoresistance of soft tissue sarcoma.
MDR phenotype induction by chemotherapeutic
agents in STS appears to be an uncommon event.
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