Transcript Sensitivity of human osteosarcomas to chemotherapy Skjalg

Sensitivity of human osteosarcomas to chemotherapy
Skjalg Bruheim, Knut Breistøl, Birgitte Smith-Sørensen, Øyvind S Bruland2, Gunhild M Maelandsmo, Øystein Fodstad1
Department of Tumor Biology, Institute for Cancer Research and Department of Radiation Oncology2, The Norwegian Radium Hospital, Oslo, Norway.
1Present address: MSB 2332, Cancer Research Institute, University of South Florida, Mobile, AL 36688-0002
Introduction
Results
Osteosarcoma is a relatively rare tumor, comprising around 0.2 % of all malignancies and most commonly affecting
children and young adults. In contrast to the situation for most solid tumors, in osteosarcoma patients chemotherapy
has a curative potential. In the absence of reliable markers that could predict the response of the individual drugs used,
patients are treated with multimodal regimens essentially including high-dose methotrexate, doxorubicin, cisplatin and
ifosfamide. Due to the aggressive treatment of osteosarcoma patients, long-term toxicity and morbidity is not
uncommon. It is likely that some patients could be cured with a simpler and less toxic treatment regimen, whereas one
third of the patients succumb to their disease without beneficial effect from current treatment options. Hence, for
major improvements in the management of osteosarcoma, a more individualised treatment as well as novel treatment
alternatives are warranted.
Around 20% of xenotransplanted osteosarcoma specimens showed take in nude mice, and gave rise to 11
established xenografts, from which parental tumor tissue was received from ten different patients. Histology and
origin of the xenografts along with the main clinical characteristics of the patients are displayed in Table 1. Seven
xenografts originate from primary tumors, whereas four were established from lung metastases. The median tumor
volume doubling time varied between 3 and 20 days. Growth curves for the individual xenografts are shown in
Figure 1.
Table 1. Clinical characteristics of osteosarcoma patients.
Immunodeficient mice and rats have contributed substantially in preclinical evaluation of anti-cancer drugs. The
xenograft models can also be in used in the search for molecular markers of drug sensitivity/resistance, and drugs of
interest can be studied individually.
Patient
characteristics
Xenograft
We have established a panel of eleven human osteosarcoma xenograft lines, grown sc in nude mice. The anti-tumor
activities of doxorubicin, cisplatin, ifosfamide methotrexate, and lomustine, as well as the expression of some genes
known to encode drug resistance, have been assessed. Microarray analysis on human osteosarcoma xenograft tissue
before and after therapy is ongoing.
Materials and methods
Table 2 summarises the antitumor activity of the various drugs and the mRNA expression of MGMT, Topo II, GST-p,
MRP-1 and MDR1. Statistical analysis revealed relatively low, but significant negative correlations between
antitumor activity of lomustine and MGMT expression, and for doxorubicin and GST-p, whereas the efficacy of
cisplatin somehow surprisingly correlated positively with MRP-1 expression (Table 3).
SBX
Xenograft
ALSKX TSXpr1 TSXpr2 HPBX
TPMX
TTX
KPDX
FTX
++++
+++
+++
+
(+)
(+)
(+)
-
-
-
++++
++
(+)
-
+++
-
-
-
++++
-
-
Cisplatin
+++
++
-
-
-
-
(+)
-
++++
-
-
Lungs, bilaterally and arm
Doxorubicine
-
+
-
-
+
-
(+)
-
++++
(+)
-
Methotrexate
+
-
-
-
-
-
-
-
-
-
-
MGMT
-
-
+++
+
+
ND
++(+)
-
-
+++
++(+)
GST-p
Resistance gene
MRP
++
-
+++
++
+
ND
++
+++
+
+++
++
++
++(+)
++(+)
++(+)
(+)
ND
++(+)
-
++(+)
-
+
MDR1
+
+
+
+
-
ND
+
+
++
++
-
TOPO II
++
+
+++
++
++
ND
++(+)
-
+++
++
+
Drug
OHS
AOX
Xenograft tissue origin
Chemotherapy prior to tissue sampling
Subsequent metastatic
3
5
Chemotherapy
Response
disease
Ifosfamide
++++
Lungs and mediastinum
Lomustine
Lungs, bilaterally
Age
Sex
HPBX
19
M
Chondroblastic
4
Tibia
Lungs
Lung metastasis
None 4
KPDX
8
M
Osteoblastic
4
Distal femur
M-
Primary
MTX
AOX
37
M
Osteoblastic
2
Femur
M-
Primary
None
ALSKX
42
M
Osteoblastic
Femur
Lungs, bilaterally
Lung metastasis
MTX, DOX, CPT
Grade II
Lungs, bilaterally
FTX
17
M
Osteoblastic
4
Femur
Lung, solitary
Lung metastasis
MTX, DOX, CPT
Grade I
Lungs
TTX
40
M
Fibroblastic
4
Os frontalis
M-
Primary
None
OHS
16
M
Osteoblastic
4
Distal femur
Skeleton, multiple
Primary
MTX, CIS-PT
SBX
68
F
4
Femur diafysis
M-
Primary
None
Lungs, abdomen
TPMX
34
M
Osteoblastic
3
Proximal femur
M-
Primary
None
Lungs, bilaterally and brain
17
M
Osteoblastic
4
Distal femur
M-
Primary
MTX, BCD
Grade II
Lungs
17
M
Osteoblastic
4
Distal femur
Lungs, bilaterally
Lung metastasis
IFO
Unknown
Lungs and spine
TSX pr.1
1
TSX pr.2
1
Histology
Tumour characteristics
Extent of disease at
Grade Primary tumour tissue sampling 2
Table 2. Anti tumor activity and expression of resistance genes
Progression
Resistance gene
Lungs, and local relapse
Progression
Skeleton
Upper panel: Summary of antitumour activities of ifosfamide, cisplatin, lomustine doxorubicine, and methotrexate
in s.c. human osteosarcoma xenografts.
Lower panel: mRNA expression of O6-Methylguanine DNA
Methyltransferase (MGMT), DNA topoisomerase II (Topo II), Gluthathione-S-Transferase (GST)-p, Multidrugresistance related protein-1 (MRP-1) and P-Glycoprotein (MDR1).
1
TSX pr.1 and TSX pr.2 were established from the primary tumor and lung metastases respectively, of the same patient. 2 M - : No metastatic disease. 3 MTX: methotrexate;
4 Received MTX and BCD preoperatively to resection of primary tumor x
DOX: doxorubicin; CPT: cisplatin; IFO: ifosfamide; BCD: bleomycin, cyclophosphamide, dactinomycin.
years earlier. 5 Response to chemotherapy according to Huvos grading of chemotherapy effects on primary osteosarcoma.
Establishment of tumour xenografts and assessment of antitumor activity
Xenografts were established by sc transplantation of fragments of tissue from biopsies or surgically removed tumours into the flanks of nude mice. For
therapy experiments, tumour fragments of about 2x2x2 mm were implanted sc, and the animals were randomised for treatment according to tumor size
when the average tumor diameter was about 6 mm.
Maximal tolerable doses (MTD), allowing a median bodyweight loss up to 15 %, were found to be 8 mg/kg of doxorubicin, 5 mg/kg of cisplatin, 150
mg/kg of methotrexate, 240 mg/kg of ifosfamide and 20 mg/kg of lomustine All drugs were administered weekly x 2, lomustine ip, whereas the other
drugs were given iv. Tumor volume was calculated according to the formula 0.5 x length x width2. Antitumor effects were assessed from calculations of
specific growth delay (SGD) and maximal growth inhibition (T/C%). Based on median relative tumor volumes (RTV), SGD and T/C% was calculated
according to the formulas:
OHS
SBX
FTX
TTX
ALSKX
KPDX
HPBX
TPMX
TSX pr.1
AOX
TSX pr.2
2500
Median RTV
SGD = (TDtreated – TDcontrol) / TDcontrol
3000
2000
Table 3. Coerrelations between gene expression and drug
efficacy (1-T/C) in human osteosarcoma xenografts.
1500
(the time for one (TD200) or two (TD400) median RTV doubling times was used to calculate values for SGD200 or SGD400)
1000
and
r
Slope
P-value
Lomustine
MGMT
-0,72
-2,64
<0,01
Cisplatin
Doxorubicin
MRP
GST-p
0,82
-0,66
3,22
-2,26
<0,005
<0,05
0
0
The antitumor activity was defined as: (+) SGD >1.0 or T/C% < 50 %; + SGD >1.0 and T/C% < 50 %; ++ SGD >1.5 and T/C% < 40 %; +++ SGD >2.0
and T/C% < 25 %; ++++ SGD >3.0 and T/C% < 10 %.
Resistance
gene
r, Pearson correlation coefficient; Slope, slope of the regression line; Only P-values for a
two sided students t-test <0,05 were regarded as significant and displayed in the table.
500
T/C% = (RTVtreated / RTVcontrol) * 100%
Drug
20
40
60
80
100
120
Days
Figure 1. Growth curves of human osteosarcoma xenografts
Assessment of mRNA expression
The mRNA expression of the drug resistance-associated genes O6-Methylguanine DNA methyltransferase (MGMT), DNA topoisomerase II (Topo II),
Glutathione-S-transferase (GST)-p, Multidrug-resistance related protein (MRP-1) and p-Glycoprotein (MDR1) were determined by Northern blot
analysis. The levels of specific RNA were calculated relative to the amount of 18s rRNA and subsequently classified as follows: -/+, undetectable/low
expression, ++ and +++, high or very high expression.
The patients, from whom the xenografted tissue originated, responded poorly or moderately to chemotherapy and all
ten succumbed to their disease. This indicates that growth in nude mice is associated with poor clinical outcome, as
others previously have shown for soft tissue sarcoma.
Statistical analysis
To estimate correlation between treatment efficacy and the mRNA expression of resistance associated genes, sample correlation coefficients were
calculated according to the formula:
r =  (Xi - Xmean)(Yi - Ymean) /  [ (Xi - Xmean)2] [(Yi - Ymean)2] ,
where Xi represented the sensitivity (1-T/C) of xenograft i to drug X and Yi represented the expression of gene Y in xenograft i. Xmean represented the mean
sensitivity to the drug X and Ymean represented the mean expression of gene Y. The test statistic t = (n-2) r / (1-r2), where d.f. = n-2 for t, was used to
test for the level of significance at which the null hypothesis H0: r = 0 could be rejected in favour of H1: r  0.
When defining drug efficacy as SGD>1 and T/C%<50, ifosfamide, lomustine, doxorubicine, cisplatin and
methotrexate were effective in 5/11, 4/11, 3/11, 3/11 and 1/10 of the xenografts, respectively. Five of the 11 tumours
were resistant to all compounds tested. For ifosfamide, cisplatin and doxorubicin, these results corroborates with
response rates seen with these drugs in the clinic. Because of the low dose-intesity achieved without lecovourin
rescue it is not supprising that methotrexate only was weakly effective in just one of the xenografts. Because
osteosarcoma patients are treated with multimodal chemotherapy, it was not possible to compare directly responses
in the individual patients with the respective xenografts.
Conclusion
We have established a well-characterised panel of human osteosarcoma xenograft lines, for use in preclinical
evaluation of new anticancer therapies. This panel gives a relatively conservative estimate of the antitumor
activity of the conventional drugs tested.
With the exception of MTX, the effects of commonly used drugs in the clinic were reflected in the xenograft
models.
The responsiveness of human osteosarcoma xenografts to doxorubicin was conversely associated with GST-p
expression, whereas the sensitivity to cisplatin surprisingly showed positive correlation to MRP-1 expression